Yaara Zwang

Loss of ATRX, Genome Instability, and an Altered DNA Damage Response Are Hallmarks of the Alternative Lengthening of Telomeres Pathway

The Alternative Lengthening of Telomeres (ALT) pathway is a telomerase-independent pathway for telomere maintenance that is active in a significant subset of human cancers and in vitro immortalized cell lines. ALT is thought to involve templated extension of telomeres through homologous recombination, but the genetic or epigenetic changes that unleash ALT are not known. Recently, mutations in the ATRX/DAXX chromatin remodeling complex and histone H3.3 were found to correlate with features of ALT in pancreatic neuroendocrine cancers, pediatric glioblastomas, and other tumors of the central nervous system, suggesting that these mutations might contribute to the activation of the ALT pathway in these cancers. We have taken a comprehensive approach to deciphering ALT by applying genomic, molecular biological, and cell biological approaches to a panel of 22 ALT cell lines, including cell lines derived in vitro. Here we show that loss of ATRX protein and mutations in the ATRX gene are hallmarks of ALT–immortalized cell lines. In addition, ALT is associated with extensive genome rearrangements, marked micronucleation, defects in the G2/M checkpoint, and altered double-strand break (DSB) repair. These attributes will facilitate the diagnosis and treatment of ALT positive human cancers.

KRAS and YAP1 converge to regulate EMT and tumor survival

Cancer cells that express oncogenic alleles of RAS typically require sustained expression of the mutant allele for survival, but the molecular basis of this oncogene dependency remains incompletely understood. To identify genes that can functionally substitute for oncogenic RAS, we systematically expressed 15,294 open reading frames in a human KRAS-dependent colon cancer cell line engineered to express an inducible KRAS-specific shRNA. We found 147 genes that promoted survival upon KRAS suppression. In particular, the transcriptional coactivator YAP1 rescued cell viability in KRAS-dependent cells upon suppression of KRAS and was required for KRAS-induced cell transformation. Acquired resistance to Kras suppression in a Kras-driven murine lung cancer model also involved increased YAP1 signaling. KRAS and YAP1 converge on the transcription factor FOS and activate a transcriptional program involved in regulating the epithelial-mesenchymal transition (EMT). Together, these findings implicate transcriptional regulation of EMT by YAP1 as a significant component of oncogenic RAS signaling.

Tyrosine phosphorylation of Mdm2 by c-Abl: implications for p53 regulation

The p53 tumor suppressor is inhibited and destabilized by Mdm2. However, under stress conditions, this downregulation is relieved, allowing the accumulation of biologically active p53. Recently we showed that c‐Abl is important for p53 activation under stress conditions. In response to DNA damage, c‐Abl protects p53 by neutralizing the inhibitory effects of Mdm2. In this study we ask whether this neutralization involves a direct interplay between c‐Abl and Mdm2, and what is the contribution of the c‐Abl kinase activity? We demonstrate that the kinase activity of c‐Abl is required for maintaining the basal levels of p53 expression and for achieving maximal accumulation of p53 in response to DNA damage. Importantly, c‐Abl binds and phosphorylates Mdm2 in vivo and in vitro. We characterize Hdm2 (human Mdm2) phosphorylation at Tyr394. Substitution of Tyr394 by Phe394 enhances the ability of Mdm2 to promote p53 degradation and to inhibit its transcriptional and apoptotic activities. Our results suggest that phosphorylation of Mdm2 by c‐Abl impairs the inhibition of p53 by Mdm2, hence defining a novel mechanism by which c‐Abl activates p53.

p38 MAP kinase mediates stress-induced internalization of EGFR: implications for cancer chemotherapy

The epidermal growth factor receptor (EGFR) frequently associates with cancer and already serves as a target for therapy. We report that inflammatory cytokines and ultraviolet (UV) irradiation respectively induce transient or sustained phosphorylation of EGFR. Subsequently, EGFR internalizes via a Clathrin‐mediated process. In cytokine‐stimulated cells, EGFR recycles back to the cell surface, whereas in irradiated cells it arrests in Rab5‐containing endosomes. Under both conditions, receptor internalization is instigated by the p38 stress‐induced kinase. The underlying mechanism entails phosphorylation of EGFR at a short segment (amino acids 1002–1022) containing multiple serines and threonines, as well as phosphorylation of two Rab5 effectors, EEA1 and GDI. Like UV irradiation, a chemotherapeutic agent activates p38 and accelerates receptor internalization. We demonstrate that abrogating EGFR internalization reduces the efficacy of chemotherapy‐induced cell death. Hence, by preventing EGFR‐mediated survival signaling, the internalization route we uncovered enhances the cytotoxic effect of drugs like cis‐platinum, which may underlie interactions between chemotherapy and EGFR‐targeting drugs.

Defective ubiquitinylation of EGFR mutants of lung cancer confers prolonged signaling.

Several distinct mutations within the kinase domain of the epidermal growth factor receptor (EGFR) are associated with non-small cell lung cancer, but mechanisms underlying their oncogenic potential are incompletely understood. Although normally ligand-induced kinase activation targets EGFR to Cbl-mediated receptor ubiquitinylation and subsequent degradation in lysosomes, we report that certain EGFR mutants escape this regulation. Defective endocytosis characterizes a deletion mutant of EGFR, as well as a point mutant (L858R-EGFR), whose association with c-Cbl and ubiquitinylation are impaired. Our data raise the possibility that refractoriness of L858R-EGFR to downregulation is due to enhanced heterodimerization with the oncogene product HER2, which leads to persistent stimulation.

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.

EGF Decreases the Abundance of MicroRNAs That Restrain Oncogenic Transcription Factors

Some cancers showed decreased abundance of a subset of EGF-regulated microRNAs, which allows the production of oncogenic transcription factors. A Loss of Restraint Growth factors, such as epidermal growth factor (EGF), bind to receptors to stimulate cell proliferation, a process critical during development and in wound healing. Dysregulation of the signaling pathways initiated by the EGF receptor (EGFR) has been implicated in cancer. Noting that aberrant expression of microRNAs, small noncoding RNAs that inhibit the expression of target genes, is common in human malignancies, Avraham et al. explored the role of microRNAs in regulating EGFR signaling. They found that EGF elicited a rapid—and transient—decrease in the abundance of a group of 23 microRNAs, thereby enabling the induction of potentially oncogenic transcription factor targets. Moreover, the abundance of this group of microRNAs was decreased in breast cancers and brain cancers with molecular lesions consistent with increased EGFR signaling. The authors conclude that, under basal conditions, this group of microRNAs restrains potentially oncogenic signaling pathways downstream of the EGFR. Their decreased abundance in cancer thus enables the dysregulated activity of oncogenic transcription factors and signaling pathways transiently activated by EGF signaling, thereby promoting the aberrant cellular behaviors associated with cancer. Epidermal growth factor (EGF) stimulates cells by launching gene expression programs that are frequently deregulated in cancer. MicroRNAs, which attenuate gene expression by binding complementary regions in messenger RNAs, are broadly implicated in cancer. Using genome-wide approaches, we showed that EGF stimulation initiates a coordinated transcriptional program of microRNAs and transcription factors. The earliest event involved a decrease in the abundance of a subset of 23 microRNAs. This step permitted rapid induction of oncogenic transcription factors, such as c-FOS, encoded by immediate early genes. In line with roles as suppressors of EGF receptor (EGFR) signaling, we report that the abundance of this early subset of microRNAs is decreased in breast and in brain tumors driven by the EGFR or the closely related HER2. These findings identify specific microRNAs as attenuators of growth factor signaling and oncogenesis.

Systems Biology of Growth Factor‐Induced Receptor Endocytosis

Clathrin‐mediated endocytosis sorts for degradation of more than 50 different growth factor receptors capable of relaying growth and differentiation signals by means of their cytoplasm‐facing, intrinsic tyrosine kinase activity. The kinetics and alternative routings of receptor endocytosis critically regulate growth factor signaling, which underscores the importance of understanding mechanisms underlying fail‐safe operation (robustness) and fidelity of the pathway. Like other robust systems, a layered hub‐centric network controls receptor endocytosis. Characteristically, the modular hubs (e.g., AP2–Eps15 and Hrs) contain a membrane‐anchoring lipid‐binding domain, an ubiquitin‐binding module, which recruits ubiquitinylated cargo, and a machinery enabling homo‐assembly. Scheduled hub transitions, as well as cascades of Rab family guanosine triphosphatases and membrane bending machineries, define points of commitment to vesicle budding, thereby securing unidirectional trafficking. System’s bistability permits stimulation by a growth factor, which oscillates a series of switches based on posttranslational protein modifications (i.e., phosphorylation, ubiquitinylation and neddylation), as well as transient low‐affinity/high‐avidity protein assemblies. Cbl family ubiquitin ligases, along with a set of phosphotyrosine‐binding adaptors (e.g., Grb2), integrate receptor endocytosis into the densely wired networks of signal transduction pathways, which are involved in health and disease.

Two Phases of Mitogenic Signaling Unveil Roles for p53 and EGR1 in Elimination of Inconsistent Growth Signals

Normal cells require continuous exposure to growth factors in order to cross a restriction point and commit to cell-cycle progression. This can be replaced by two short, appropriately spaced pulses of growth factors, where the first pulse primes a process, which is completed by the second pulse, and enables restriction point crossing. Through integration of comprehensive proteomic and transcriptomic analyses of each pulse, we identified three processes that regulate restriction point crossing: (1) The first pulse induces essential metabolic enzymes and activates p53-dependent restraining processes. (2) The second pulse eliminates, via the PI3K/AKT pathway, the suppressive action of p53, as well as (3) sets an ERK-EGR1 threshold mechanism, which digitizes graded external signals into an all-or-none decision obligatory for S phase entry. Together, our findings uncover two gating mechanisms, which ensure that cells ignore fortuitous growth factors and undergo proliferation only in response to consistent mitogenic signals.

Potential role of intratumor bacteria in mediating tumor resistance to the chemotherapeutic drug gemcitabine

In model systems, bacteria present in human pancreatic tumors confer resistance to the anticancer drug gemcitabine. Debugging a cancer therapy Microbes contribute not only to the development of human diseases but also to the response of diseases to treatment. Geller et al. show that certain bacteria express enzymes capable of metabolizing the cancer chemotherapeutic drug gemcitabine into an inactive form. When bacteria were introduced into tumors growing in mice, the tumors became resistant to gemcitabine, an effect that was reversed by antibiotic treatment. Interestingly, a high percentage of human pancreatic ductal adenocarcinomas, a tumor type commonly treated with gemcitabine, contain the culprit bacteria. These correlative results raise the tantalizing possibility that the efficacy of an existing therapy for this lethal cancer might be improved by cotreatment with antibiotics. Science, this issue p. 1156 Growing evidence suggests that microbes can influence the efficacy of cancer therapies. By studying colon cancer models, we found that bacteria can metabolize the chemotherapeutic drug gemcitabine (2′,2′-difluorodeoxycytidine) into its inactive form, 2′,2′-difluorodeoxyuridine. Metabolism was dependent on the expression of a long isoform of the bacterial enzyme cytidine deaminase (CDDL), seen primarily in Gammaproteobacteria. In a colon cancer mouse model, gemcitabine resistance was induced by intratumor Gammaproteobacteria, dependent on bacterial CDDL expression, and abrogated by cotreatment with the antibiotic ciprofloxacin. Gemcitabine is commonly used to treat pancreatic ductal adenocarcinoma (PDAC), and we hypothesized that intratumor bacteria might contribute to drug resistance of these tumors. Consistent with this possibility, we found that of the 113 human PDACs that were tested, 86 (76%) were positive for bacteria, mainly Gammaproteobacteria.