A small epitope shared by p53 and an unrelated protein upregulated after adenovirus infection.

Cancers commonly harbor point mutations in TP53 that cause overexpression of functionally inactive p53 proteins. These mutant forms of p53 are immunogenic, and therefore present tantalizing targets for new forms of immunotherapy. Understanding how the immune system recognizes p53 is an important prerequisite for the development of targeted therapeutic strategies designed to exploit this common neoantigen. Monoclonal antibodies have been extensively used to probe the structural conformation of the varied isoforms of p53 and their respective mutants, and are still indispensable tools for studying the complex biological functions of these proteins. In this report, we describe the mapping of a novel epitope on p53 that appears to be shared by heat shock proteins (HSPs), which are typically upregulated in response to a variety of viral infections.

AdenoBuilder: A platform for the modular assembly of recombinant adenoviruses.

The AdenoBuilder platform enables the in vitro assembly of recombinant vectors from plasmid inserts that span the adenovirus genome. Two advantages of AdenoBuilder are the ease of modifying the genome and the ability to produce multicomponent vectors in a single step, facilitating parallel approaches to vector optimization. This protocol describes how to introduce transgenes in place of the endogenous Human Adenovirus serotype 5 (HAd5) E1 and/or E3 genes and can be applied to other parts of the HAd5 genome. For complete details on the use and execution of this protocol, please refer to Miciak et al. (2018).

Preventing collateral damage.

In pigs, nitrate supplements can protect salivary glands from the damage caused by radiation therapy to the head and neck.

EMT and Back Again: Visualizing the Dynamic Phenotypes of Metastasis.

To what extent does the acquisition of mesenchymal phenotypes by tumor epithelial cells contribute to metastasis? A definitive answer to this question has remained elusive despite much experimentation and debate. Recently, an influential study based on fluorescence-based lineage tracing technology provided evidence that very few of the cells that populate experimental metastases in fact undergo the epithelial-to-mesenchymal transition. Persistent questions regarding the concordance between marker conversion and cell phenotypes prompted Lourenco and colleagues to complement their lineage tracking system with single-cell analysis. This granular approach provides an unprecedented view of the phenotypic transitions that take place during metastasis and their striking heterogeneity.See related article by Lourenco et al., p. 163.

Increased uptake of doxorubicin by cells undergoing heat stress does not explain its synergistic cytotoxicity with hyperthermia.

Purpose: A proposed mechanism for the enhanced effectiveness of hyperthermia and doxorubicin (Dox) combinations is increased intracellular Dox concentrations resulting from heat-induced cell stress. The purpose of this study was to determine whether specific varied Dox and heat combinations produce measurable effects greater than the additive combination, and whether these effects can be attributed to heat-induced increases in intracellular Dox concentrations. Methods: HCT116, HT29 and CT26 cells were exposed to Dox and water bath heating independently. A clonogenic survival assay was used to determine cell killing and intracellular Dox concentrations were measured in HCT116 cells with mass spectrometry. Cells were exposed to heating at 42 °C (60 min) and 0.5 µg/ml of Dox at varying intervals. Synergy was determined by curve-fitting and isobologram analysis. Results: All cell lines displayed synergistic effects of combined heating and Dox. A maximum synergistic effect was achieved with simultaneous cell exposure to Dox and heat. For exposures at 42 °C, the synergistic effect was most pronounced at Dox concentrations <0.5 µg/ml. Increased intracellular concentrations of Dox in HCT116 cells caused by heat-stress did not generate a concomitant thermal enhancement. Conclusions: Simultaneous exposure of HCT116 cells to heating and Dox is more effective than sequential exposure. Heat-induced cell responses are accompanied by increased intracellular Dox concentrations; however, clonogenic survival data do not support this as the cause for synergistic cytotoxicity.

Seamless assembly of recombinant adenoviral genomes from high-copy plasmids.

The adenoviruses are essential tools for basic research and therapeutic development. Robust methods for the generation of mutant and recombinant viruses are crucial for these diverse applications. Here we describe a simple plasmid-based method that permits highly efficient modification of the adenoviral genome and rapid production of high-titer virus stocks. The 36-kilobase genome of adenovirus serotype 5 was divided into seven tractable blocks that could be individually modified in a single step and reassembled in vitro. Because the system is composed of compact modules, modifications at different loci can be readily recombined. Viral assemblies were delivered to packaging cells by electroporation, a strategy that consistently resulted in the de novo production of 108 infectious units in 3-5 days. In principle, a similar strategy could be applied to any other adenovirus serotype or to other double-strand DNA viruses.

Tumor-associated APE1 variant exhibits reduced complementation efficiency but does not promote cancer cell phenotypes.

Base excision repair (BER) is the major pathway for coping with most forms of endogenous DNA damage, and defects in the process have been associated with carcinogenesis. Apurinic/apyrimidinic endonuclease 1 (APE1) is a central participant in BER, functioning as a critical endonuclease in the processing of noncoding abasic sites in DNA. Evidence has suggested that APE1 missense mutants, as well as altered expression or localization of the protein, can contribute to disease manifestation. We report herein that the tumor-associated APE1 variant, R237C, shows reduced complementation efficiency of the methyl methanesulfonate hypersensitivity and impaired cell growth exhibited by APE1-deficient mouse embryonic fibroblasts. Overexpression of wild-type APE1 or the R237C variant in the nontransformed C127I mouse cell line had no effect on proliferation, cell cycle status, steady-state DNA damage levels, mitochondrial function, or cellular transformation. A human cell line heterozygous for an APE1 knockout allele had lower levels of endogenous APE1, increased cellular sensitivity to DNA-damaging agents, impaired proliferation with time, and a distinct global gene expression pattern consistent with a stress phenotype. Our results indicate that: (i) the tumor-associated R237C variant is a possible susceptibility factor, but not likely a driver of cancer cell phenotypes, (ii) overexpression of APE1 does not readily promote cellular transformation, and (iii) haploinsufficiency at the APE1 locus can have profound cellular consequences, consistent with BER playing a critical role in proliferating cells. Environ. Mol. Mutagen. 58:84-98, 2017. © 2017 Wiley Periodicals, Inc.

Long story short: p53 mediates innate immunity.

The story of p53 and how we came to understand it is punctuated by fundamental insights into the essence of cancer. In the decades since its discovery, p53 has been shown to be centrally involved in most, if not all, of the cellular processes that maintain tissue homeostasis. Extensive functional analyses of p53 and its tumor-associated mutants have illuminated many of the common defects shared by most cancer cells. As the central character in a tale that continues to unfold, p53 has become increasingly familiar and yet remains surprisingly inscrutable. New relationships periodically come to light, and surprising, novel activities continue to emerge, thereby revealing new dimensions and aspects of its function. What lies at the very core of this complex protagonist? What is its prime motivation? As every avid reader knows, the elements of character are profoundly shaped by adversity--originating from within and without. And so it is with p53. This review will briefly recap the coordinated responses of p53 to viral infection, and outline a hypothetical model that would explain how an abundance of seemingly unrelated phenotypic attributes may in the end reflect a singular function. All stories eventually draw to a conclusion. This epic tale may eventually leave us with the realization that p53, most simply described, is a protein that evolved to mediate immune surveillance.

Pathways to chromothripsis.

Chromothripsis is a recently recognized mode of genetic instability that generates chromosomes with strikingly large numbers of segmental re-arrangements. While the characterization of these derivative chromosomes has provided new insights into the processes by which cancer genomes can evolve, the underlying signaling events and molecular mechanisms remain unknown. In medulloblastomas, chromothripsis has been observed to occur in the context of mutational inactivation of p53 and activation of the canonical Hedgehog (Hh) pathway. Recent studies have illuminated mechanistic links between these 2 signaling pathways, including a novel PTCH1 homolog that is regulated by p53. Here, we integrate this new pathway into a hypothetical model for the catastrophic DNA breakage that appears to trigger profound chromosomal rearrangements.

Effective treatment of diverse medulloblastoma models with mebendazole and its impact on tumor angiogenesis.

BACKGROUND: Medulloblastoma is the most common malignant brain tumor in children. Current standard treatments cure 40%-60% of patients, while the majority of survivors suffer long-term neurological sequelae. The identification of 4 molecular groups of medulloblastoma improved the clinical management with the development of targeted therapies; however, the tumor acquires resistance quickly. Mebendazole (MBZ) has a long safety record as antiparasitic in children and has been recently implicated in inhibition of various tyrosine kinases in vitro. Here, we investigated the efficacy of MBZ in various medulloblastoma subtypes and MBZ's impact on vascular endothelial growth factor receptor 2 (VEGFR2) and tumor angiogenesis. METHODS: The inhibition of MBZ on VEGFR2 kinase was investigated in an autophosphorylation assay and a cell-free kinase assay. Mice bearing orthotopic PTCH1-mutant medulloblastoma allografts, a group 3 medulloblastoma xenograft, and a PTCH1-mutant medulloblastoma with acquired resistance to the smoothened inhibitor vismodegib were treated with MBZ. The survival benefit and the impact on tumor angiogenesis and VEGFR2 kinase function were analyzed. RESULTS: We determined that MBZ interferes with VEGFR2 kinase by competing with ATP. MBZ selectively inhibited tumor angiogenesis but not the normal brain vasculatures in orthotopic medulloblastoma models and suppressed VEGFR2 kinase in vivo. MBZ significantly extended the survival of medulloblastoma models derived from different molecular backgrounds. CONCLUSION: Our findings support testing of MBZ as a possible low-toxicity therapy for medulloblastomas of various molecular subtypes, including tumors with acquired vismodegib resistance. Its antitumor mechanism may be partially explained by inhibition of tumor angiogenesis.

Repurposing the antihelmintic mebendazole as a hedgehog inhibitor.

The hedgehog (Hh) signaling pathway is activated in many types of cancer and therefore presents an attractive target for new anticancer agents. Here, we show that mebendazole, a benzamidazole with a long history of safe use against nematode infestations and hydatid disease, potently inhibited Hh signaling and slowed the growth of Hh-driven human medulloblastoma cells at clinically attainable concentrations. As an antiparasitic, mebendazole avidly binds nematode tubulin and causes inhibition of intestinal microtubule synthesis. In human cells, mebendazole suppressed the formation of the primary cilium, a microtubule-based organelle that functions as a signaling hub for Hh pathway activation. The inhibition of Hh signaling by mebendazole was unaffected by mutants in the gene that encodes human Smoothened (SMO), which are selectively propagated in cell clones that survive treatment with the Hh inhibitor vismodegib. Combination of vismodegib and mebendazole resulted in additive Hh signaling inhibition. Because mebendazole can be safely administered to adults and children at high doses over extended time periods, we propose that mebendazole could be rapidly repurposed and clinically tested as a prospective therapeutic agent for many tumors that are dependent on Hh signaling.

A PTCH1 homolog transcriptionally activated by p53 suppresses Hedgehog signaling.

The p53-mediated responses to DNA damage and the Hedgehog (Hh) signaling pathway are each recurrently dysregulated in many types of human cancer. Here we describe PTCH53, a p53 target gene that is homologous to the tumor suppressor gene PTCH1 and can function as a repressor of Hh pathway activation. PTCH53 (previously designated PTCHD4) was highly responsive to p53 in vitro and was among a small number of genes that were consistently expressed at reduced levels in diverse TP53 mutant cell lines and human tumors. Increased expression of PTCH53 inhibited canonical Hh signaling by the G protein-coupled receptor SMO. PTCH53 thus delineates a novel, inducible pathway by which p53 can repress tumorigenic Hh signals.

Polo-like kinase 2 activates an antioxidant pathway to promote the survival of cells with mitochondrial dysfunction.

We previously reported that Polo-like kinase 2 (PLK2) is highly expressed in cells with defective mitochondrial respiration and is essential for their survival. Although PLK2 has been widely studied as a cell cycle regulator, we have uncovered an antioxidant function for this kinase that activates the GSK3-NRF2 signaling pathway. Here, we report that the expression of PLK2 is responsive to oxidative stress and that PLK2 mediates antioxidant signaling by phosphorylating GSK3, thereby promoting the nuclear translocation of NRF2. We further show that the antioxidant activity of PLK2 is essential for preventing p53-dependent necrotic cell death. Thus, the regulation of redox homeostasis by PLK2 promotes the survival of cells with dysfunctional mitochondria, which may have therapeutic implications for cancer and neurodegenerative diseases.

Transcriptomes and shRNA suppressors in a TP53 allele-specific model of early-onset colon cancer in African Americans.

UNLABELLED: African Americans are disproportionately affected by early-onset, high-grade malignancies. A fraction of this cancer health disparity can be explained by genetic differences between individuals of African or European descent. Here the wild-type Pro/Pro genotype at the TP53Pro72Arg (P72R) polymorphism (SNP: rs1042522) is more frequent in African Americans with cancer than in African Americans without cancer (51% vs. 37%), and is associated with a significant increase in the rates of cancer diagnosis in African Americans. To test the hypothesis that Tp53 allele-specific gene expression may contribute to African American cancer disparities, TP53 hemizygous knockout variants were generated and characterized in the RKO colon carcinoma cell line, which is wild type for TP53 and heterozygous at the TP53Pro72Arg locus. Transcriptome profiling, using RNAseq, in response to the DNA-damaging agent etoposide revealed a large number of Tp53-regulated transcripts, but also a subset of transcripts that were TP53Pro72Arg allele specific. In addition, a shRNA-library suppressor screen for Tp53 allele-specific escape from Tp53-induced arrest was performed. Several novel RNAi suppressors of Tp53 were identified, one of which, PRDM1ß (BLIMP-1), was confirmed to be an Arg-specific transcript. Prdm1ß silences target genes by recruiting H3K9 trimethyl (H3K9me3) repressive chromatin marks, and is necessary for stem cell differentiation. These results reveal a novel model for African American cancer disparity, in which the TP53 codon 72 allele influences lifetime cancer risk by driving damaged cells to differentiation through an epigenetic mechanism involving gene silencing. IMPLICATIONS: TP53 P72R polymorphism significantly contributes to increased African American cancer disparity.

A loss-of-function mutation in PTCH1 suggests a role for autocrine hedgehog signaling in colorectal tumorigenesis.

Hedgehog (Hh) signaling is largely suppressed in the normal differentiated tissues of the adult but activated in many cancers. The Hh pathway can either be activated by the expression of Hh ligands, or by mutations that cause constitutive, ligand-independent signaling. Colorectal cancer cells frequently express Hh ligands that are believed to exert paracrine effects on the stromal component of the tumor. Evidence for a more direct role of Hh signaling on the growth and evolution of colorectal cancer cell clones has been lacking. Here, we report a loss-of-function mutation of PTCH1, a tumor suppressor in the Hh pathway, in a colorectal cancer that exhibits transcriptional upregulation of the downstream Hh gene GLI1. This finding demonstrates that autocrine Hh signaling can provide a selective advantage to evolving tumors that arise in the colorectal epithelia, and suggests a definable group of colorectal cancer patients that could derive enhanced benefit from Hh pathway inhibitors.

Genetic disruption of USP9X sensitizes colorectal cancer cells to 5-fluorouracil.

The X-linked deubiquitinase USP9X affects the stability and activity of numerous regulatory proteins that influence cell survival. Recent studies suggest that decreased USP9X expression can confer a selective advantage onto developing cancer cells and thereby promotes disease progression. To examine the effect of USP9X on the cellular responses to anticancer therapies, we derived cancer cell lines in which the USP9X locus was disrupted by homologous recombination. The resulting USP9X-deficient cancer cells exhibited increased activation of apoptotic pathways and markedly decreased clonogenic survival in response to 5-fluorouracil, a chemotherapeutic drug that is widely used for treatment of gastrointestinal malignancies. These unexpected results suggest that cancers with low USP9X expression might be specifically sensitized to some conventional therapeutic agents.

Chk1 suppresses bypass of mitosis and tetraploidization in p53-deficient cancer cells.

Many cancer cells are unable to maintain a numerically stable chromosome complement. It is well established that aberrant cell division can generate progeny with increased ploidy, but the genetic factors required for maintenance of diploidy are not well understood. Using an isogenic model system derived by gene targeting, we examined the role of Chk1 in p53-proficient and -deficient cancer cells. Targeted inactivation of a single CHK1 allele in stably diploid cells caused an elevated frequency of mitotic bypass if p53 was naturally mutated or experimentally disrupted by homologous recombination. CHK1-haploinsufficient, p53-deficient cells frequently underwent sequential rounds of DNA synthesis without an intervening mitosis. These aberrant cell cycles resulted in whole-genome endoreduplication and tetraploidization. The unscheduled bypass of mitosis could be suppressed by targeted reversion of a p53 mutation or by exogenous expression of Cdk1. In contrast, the number of tetraploid cells was not increased in isogenic cell populations that harbor hypomorphic ATR mutations, suggesting that suppression of unscheduled mitotic bypass is a distinct function of Chk1. These results are consistent with a recently described role for Chk1 in promoting the expression of genes that promote cell cycle transitions and demonstrate how Chk1 might prevent tetraploidization during the cancer cell cycle.

Targeted mutations in the ATR pathway define agent-specific requirements for cancer cell growth and survival.

Many anticancer agents induce DNA strand breaks or cause the accumulation of DNA replication intermediates. The protein encoded by ataxia-telangiectasia mutated and Rad 3-related (ATR) generates signals in response to these altered DNA structures and activates cellular survival responses. Accordingly, ATR has drawn increased attention as a potential target for novel therapeutic strategies designed to potentiate the effects of existing drugs. In this study, we use a unique panel of genetically modified human cancer cells to unambiguously test the roles of upstream and downstream components of the ATR pathway in the responses to common therapeutic agents. Upstream, the S-phase-specific cyclin-dependent kinase (Cdk) 2 was required for robust activation of ATR in response to diverse chemotherapeutic agents. While Cdk2-mediated ATR activation promoted cell survival after treatment with many drugs, signaling from ATR directly to the checkpoint kinase Chk1 was required for survival responses to only a subset of the drugs tested. These results show that specifically inhibiting the Cdk2/ATR/Chk1 pathway via distinct regulators can differentially sensitize cancer cells to a wide range of therapeutic agents.