Fred Bunz

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

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.

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. Mol Cancer Ther; 14(1); 3–13. ©2014 AACR.

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.

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. Mol Cancer Ther; 11(1); 98–107. ©2011 AACR.

The protein kinase encoded by the ataxia telangiectasia and Rad3-related (ATR) gene is activated by DNA-damaging agents that are frequently used as anticancer therapeutics. Inhibition of ATR expression in cultured cancer cells has been demonstrated to increase sensitivity to chemotherapeutic drugs, including the DNA-crosslinking agent cisplatin. Cisplatin is a widely used and effective drug, but its use is associated with significant toxicity. Here, we demonstrate that genetic inhibition of ATR expression selectively enhanced cisplatin sensitivity in human colorectal cancer cells with inactivated p53. A knock-in strategy was used to restore wild-type p53 in cells harboring wild-type or mutant ATR alleles. Knock-in of functional p53 in ATR-deficient cells restored checkpoint function, suppressed apoptotic pathways and markedly increased clonogenic survival after cisplatin treatment. These results suggest that a strategy that combines specific inhibitors of ATR and conventional therapies might promote synthetic lethality in p53-deficient tumors, and thus minimize toxicity to normal tissues.

DNA damage impairs cell growth by delaying or preventing critical processes such as DNA replication and chromosome segregation. In normal proliferating cells, initiation of these processes is controlled by genetically-defined pathways known as checkpoints. Tumors often acquire mutations that disable checkpoints and cancer cells can therefore progress unimpeded into S-phase, through G2 and into mitosis with chromosomal DNA damage. Checkpoint bypass in cancer cells is associated with cell death and loss of proliferative capacity and therefore is believed to contribute to the efficacy of DNA-damaging therapies. Are cancer cell clones that bypass checkpoints invariably more sensitive to DNA damage than checkpoint-proficient cells in normal tissues? We present evidence that the inherent survival of damaged human cells can be surprisingly independent of checkpoint control.

The activation of phase-specific cyclin-dependent kinases (Cdks) is associated with ordered cell cycle transitions. Among the mammalian Cdks, only Cdk1 is essential for somatic cell proliferation. Cdk1 can apparently substitute for Cdk2, Cdk4, and Cdk6, which are individually dispensable in mice. It is unclear if all functions of non-essential Cdks are fully redundant with Cdk1. Using a genetic approach, we show that Cdk2, the S-phase Cdk, uniquely controls the G₂/M checkpoint that prevents cells with damaged DNA from initiating mitosis. CDK2-nullizygous human cells exposed to ionizing radiation failed to exclude Cdk1 from the nucleus and exhibited a marked defect in G₂/M arrest that was unmasked by the disruption of P53. The DNA replication licensing protein Cdc6, which is normally stabilized by Cdk2, was physically associated with the checkpoint regulator ATR and was required for efficient ATR-Chk1-Cdc25A signaling. These findings demonstrate that Cdk2 maintains a balance of S-phase regulatory proteins and thereby coordinates subsequent p53-independent G₂/M checkpoint activation. Metazoan cells contain multiple Cdks that regulate cell cycle progression. Recent studies have shown that mouse cells can grow normally with just Cdk1. The roles of the non-essential Cdks remain a fundamental question. In this study, we describe the generation and detailed characterization of CDK2-knockout human somatic cells. Our study demonstrates that Cdk2 is required for robust DNA damage checkpoint signaling. Loss of Cdk2 caused a marked deficiency in the G₂/M arrest—a basic response to DNA damage—in cells that were also nullizygous for P53. We propose that the multiple Cdks present in metazoan cells provide a mechanism by which the cell cycle can be efficiently halted after DNA damage. Significantly, this study reveals a heretofore unrecognized dependence for Cdk2 in p53-deficient cancer cells.