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Cancers commonly harbor point mutations inTP53that 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.

PDF file - 74K, Effect of CDK2 on DNA replication fork dynamics. Replicating DNA was sequentially labeled with CIdU (red) and IdU (green), and the length of double-labeled DNA fibers were measured via confocal microscopy. (A) Distribution of fork rates in unperturbed HCT116 cells and the CDK2-/- derivative cell line. (B) Average fork rates in HCT116 parental and CDK2-/- cells. ** denotes significance (One-tailed paired Student's t-test, p

XLSX file - 5764K, S1. TP53 Codon 72 Polymorphism Genotype Frequency. S2. RNA-Seq Data. S3. TP53 Dependent Genes. S4. TP53 Codon 72 Dependent Genes. S5. Deep Sequencing. S6. TP53 Dependent shRNA. S7. Arg Dependent shRNA. S8. Pro Dependent shRNA.

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, PRDM1β (BLIMP-1), was confirmed to be an Arg-specific transcript. Prdm1β 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. Mol Cancer Res; 12(7); 1029–41. ©2014 AACR.

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.

Supplementary Figure 1: (A) Survival of mice with DAOY-derived orthotopic tumors at 25 mg/kg MBZ (n=4) or mock-treated (n=3). One mouse from the mock treatment group did not develop symptoms of a growing brain tumor, and was euthanized after surviving for more than six months. This mouse was not found to have a brain tumor, and was therefore omitted from this analysis. (B) RNA was harvested from untreated and treated tumors and assessed by qRT-PCR for expression of GLI1, PTCH1, and PTCH2.

Supplementary Table 1 from RNAi-Mediated Silencing of Nuclear Factor Erythroid-2–Related Factor 2 Gene Expression in Non–Small Cell Lung Cancer Inhibits Tumor Growth and Increases Efficacy of Chemotherapy

The human body is composed of about 200 different types of cells that organize into a multitude of tissues. Cancers can arise from nearly all of these cell populations. What we casually refer to as cancer is actually a diverse spectrum of human diseases. The most common cancers in adults are carcinomas, derived from epithelial cells that line body cavities and glands. Sarcomas arise from the mesenchymal tissues that form the connective tissues of the body. Melanomas, retinoblastomas, neuroblastomas and glioblastomas are derived from dividing cells in the ocular retina, immature peripheral neurons and neural support cells known as glia, respectively. Lymphomas and leukemias, sometimes referred to as the ‘liquid tumors’, arise in the tissues that give rise to lymphoid and blood cells. All of these diseases will be collectively referred to as ‘cancer’ throughout this book. The rationale for this simplification is that all of these diverse diseases have a single root cause.

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

The diverse applications of mutant and recombinant adenoviruses demand facile methods for their generation. This protocol allows the user to rapidly assemble customized adenoviral genomes from high-copy plasmids in a two-step isothermal reaction. Following in vitro assembly, full-length genomes can be directly packaged in HEK293 cells. Crude preparations of infectious virus can be prepared in less than one week.

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.

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.

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 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.

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 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.

Medulloblastoma, the most common type of pediatric brain malignancy, is an aggressive primitive neuroectodermal tumor arising from the cerebellum. The incidence peaks twice during childhood: at 3–4 years of age and at 8–9 years of age. Despite an increased molecular understanding and significant improvements in therapy, only slightly more than half of children with the diagnosis of medulloblastoma survive >10 years. Current treatments combining surgery, radiotherapy, and antiproliferative chemotherapy have been successful, but these treatments can have long-lasting adverse effects, including impaired neural development.1 In particular, radiation therapy causes late effects such as neurocognitive deficiencies, hormone deficits, and growth impairment.1 The most common chemotherapy-related late effects are ototoxicity caused by platinum drugs, secondary leukemia, and infertility following exposure to alkylating agents.1 Since the present therapy does not address about 40% of patients who still succumb to the disease,2 and the surviving patients risk severe side effects, less toxic and more efficacious therapy is needed. Recent studies have demonstrated that medulloblastoma comprises several molecularly distinct groups based on the specific genetic alterations and on the gene expression profiles. The Wnt group is typified by a gene expression signature of activated Wnt signaling, and a sonic hedgehog (SHH) group is defined by the expression of genes downstream of the hedgehog (Hh) pathway, by mutations in PTCH1, the Hh pathway tumor suppressor, and by mutations in p53. Group 3 has the worst prognosis and is often associated with MYC amplification and overexpression, while group 4 medulloblastoma presents an intermediate prognosis and sometimes is associated with cyclin-dependent kinase (CDK)6 or MYCN amplifications.2,3 The molecular or genetic basis of certain medulloblastoma groups may sensitize them to targeted therapy. This was demonstrated by the effectiveness of the Hh-pathway inhibitor vismodegib (GDC-0449), an orally bioavailable small-molecule inhibitor of smoothened (SMO), on a patient with a disseminated SHH group medulloblastoma.4 Unfortunately, in this patient, prolonged treatment with vismodegib eventually failed, as recurrent tumor had acquired mutations in SMO.4,5 This case study illustrates the risk of targeting a mutable onco-pathway and a genetically diverse tumor cell population. Compared with other solid tumors, brain tumors are especially difficult to treat, owing mainly to insufficient drug delivery to intracranial tumors. Many chemotherapeutics that are effective against brain tumor cells in vitro fail to reach effective concentrations in the intracranial tumor.6 In the pediatric patient population, drug-related neural toxicity to the developing brain further limits options. Mebendazole (MBZ) is an antiparasitic drug with a long history of safe human use against helminthic infections, even at high doses. Recently, we discovered MBZ as a potent drug in treating orthotopic glioma in mice with low toxicity.7 GBM is a highly aggressive brain tumor, and MBZ was able to significantly improve the animals' survival. MBZ is a benzimidazole, with a low molecular weight of 295 daltons and a high degree of lipophilicity contributing to brain permeability.8 MBZ binds to tubulin and disrupts the formation of microtubules that are essential in a broad range of cellular functions, such as the formation of mitotic spindle and cytoskeleton. In recent years, several mechanisms have been proposed for MBZ's antitumor activity, including B-cell lymphoma 2 inactivation and downregulation of X-linked inhibitor of apoptosis protein.9,10 We have previously reported that MBZ disrupts microtubules in glioma cells and MBZ can be efficacious in intracranial gliomas with oral administration.7 Even though tubulin is a well-known anticancer target, further details of the molecular mechanism of MBZ's antitumor activity in brain tumors have not yet been determined. Recently, Dakshanamurthy and colleagues11 applied a computational proteo-chemometric method with a library of FDA-approved compounds and identified MBZ as one potential inhibitor of vascular endothelial growth factor receptor 2 (VEGFR2), which suggested a possible role of MBZ in interfering with tumor angiogenesis. In this study, we characterized the inhibition of VEGFR2 kinase by MBZ and demonstrated that MBZ exhibited an anti-angiogenic effect and interfered with VEGFR2 activity in multiple medulloblastoma models. We showed that MBZ extended survival in intracranial models of the SHH group and group 3 medulloblastoma.

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.

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, PRDM1β (BLIMP-1), was confirmed to be an Arg-specific transcript. Prdm1β 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. Mol Cancer Res; 12(7); 1029–41. ©2014 AACR.

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.

The identification of novel approaches to specifically target the DNA-damage checkpoint response in chemotherapy-resistant cancer stem cells (CSC) of solid tumors has recently attracted great interest. We show here in colon cancer cell lines and primary colon cancer cells that inhibition of checkpoint-modulating phosphoinositide 3-kinase-related (PIK) kinases preferentially depletes the chemoresistant and exclusively tumorigenic CD133+ cell fraction. We observed a time- and dose-dependent disproportionally pronounced loss of CD133+ cells and the consecutive lack of in vitro and in vivo tumorigenicity of the remaining cells. Depletion of CD133+ cells was initiated through apoptosis of cycling CD133+ cells and further substantiated through subsequent recruitment of quiescent CD133+ cells into the cell cycle followed by their elimination. Models using specific PIK kinase inhibitors, somatic cell gene targeting, and RNA interference demonstrated that the observed detrimental effects of caffeine on CSC were attributable specifically to the inhibition of the PIK kinase ataxia telangiectasia- and Rad3-related (ATR). Mechanistically, phosphorylation of CHK1 checkpoint homolog (S. pombe; CHK1) was significantly enhanced in CD133+ as compared with CD133− cells on treatment with DNA interstrand-crosslinking (ICL) agents, indicating a preferential activation of the ATR/CHK1-dependent DNA-damage response in tumorigenic CD133+ cells. Consistently, the chemoresistance of CD133+ cells toward DNA ICL agents was overcome through inhibition of ATR/CHK1-signaling. In conclusion, our study illustrates a novel target to eliminate the tumorigenic CD133+ cell population in colon cancer and provides another rationale for the development of specific ATR-inhibitors.

The protein kinase encoded by the ataxia-telangiectasia and Rad3-related (ATR) gene is activated by DNA damaging agents that are frequently employed 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 employed 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 knockin strategy was employed to restore wild type p53 in cells harboring wild type or mutant ATR alleles. Knockin of functional p53 in ATR-deficient cells restored checkpoint function, suppressed apoptotic pathways, and dramatically 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 while minimizing 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.

Through targeted homologous recombination, we developed a panel of matched colorectal cancer cell lines that differ only with respect to their endogenousTP53status. We then used these lines to define the genes whose expression was altered after DNA damage induced by ionizing radiation. Transcriptome analyses revealed a consistent up-regulation of polo-like kinase 1 (PLK1) as well as other genes controlling the G2/M transition in the cells whoseTP53genes were inactivated compared with those with WTTP53genes. This led to the hypothesis that the viability of stressed cells without WTTP53depended onPLK1. This hypothesis was validated by demonstrating that stressed cancer cells without WTTP53alleles were highly sensitive to PLK1 inhibitors, both in vivo and in vitro.

Chk1 is widely known as a DNA damage checkpoint signaling protein. Unlike many other checkpoint proteins, Chk1 also plays an essential but poorly defined role in the proliferation of unperturbed cells. Activation of Chk1 after DNA damage is known to require the phosphorylation of several C-terminal residues, including the highly conserved S317 and S345 sites. To evaluate the respective roles of these individual sites and assess their contribution to the functions of Chk1, we used a gene targeting approach to introduce point mutations into the endogenous human CHK1 locus. We report that the essential and nonessential functions of Chk1 are regulated through distinct phosphorylation events and can be genetically uncoupled. The DNA damage response function of Chk1 was nonessential. Targeted mutation of S317 abrogated G 2 /M checkpoint activation, prevented subsequent phosphorylation of Chk1, impaired efficient progression of DNA replication forks, and increased fork stalling, but did not impact viability. Thus, the nonessential DNA damage response function of Chk1 could be unambiguously linked to its role in DNA replication control. In contrast, a CHK1 allele with mutated S345 did not support viability, indicating an essential role for this residue during the unperturbed cell cycle. A distinct, physiologic mode of S345 phosphorylation, initiated at the centrosome during unperturbed mitosis was independent of codon 317 status and mechanistically distinct from the ordered and sequential phosphorylation of serine residues on Chk1 induced by DNA damage. Our findings suggest an essential regulatory role for Chk1 phosphorylation during mitotic progression.

Bloom syndrome is caused by homozygous mutations in BLM, which encodes a RecQ DNA helicase. Patient-derived cells deficient in BLM helicase activity exhibit genetic instability - apparent cytogenetically as sister chromatid exchanges - and activated DNA damage signaling. In this report, we show that BLM-knockout colorectal cancer cells exhibited endogenous, ATM-dependent double-strand DNA break responses similar to those recently observed in Bloom syndrome patient-derived cells. Xenograft tumors established from BLM-deficient cancer cells were not radiosensitive, but exhibited growth impairment that was comparable to that of wild type tumors treated with a single, high dose of ionizing radiation. These results suggest that pharmacological inhibitors of BLM would have a radiomimetic effect, and that transient inhibition of BLM activity might be a viable strategy for anticancer therapy.

Nuclear factor erythroid-2–related factor 2 (Nrf2) is a redox-sensitive transcription factor that regulates the expression of electrophile and xenobiotic detoxification enzymes and efflux proteins, which confer cytoprotection against oxidative stress and apoptosis in normal cells. Loss of function mutations in the Nrf2 inhibitor, Kelch-like ECH-associated protein (Keap1), results in constitutive activation of Nrf2 function in non–small cell lung cancer. In this study, we show that constitutive activation of Nrf2 in lung cancer cells promotes tumorigenicity and contributes to chemoresistance by up-regulation of glutathione, thioredoxin, and the drug efflux pathways involved in detoxification of electrophiles and broad spectrum of drugs. RNAi-mediated reduction of Nrf2 expression in lung cancer cells induces generation of reactive oxygen species, suppresses tumor growth, and results in increased sensitivity to chemotherapeutic drug–induced cell death in vitro and in vivo. Inhibiting Nrf2 expression using naked siRNA duplexes in combination with carboplatin significantly inhibits tumor growth in a subcutaneous model of lung cancer. Thus, targeting Nrf2 activity in lung cancers, particularly those with Keap1 mutations, could be a promising strategy to inhibit tumor growth and circumvent chemoresistance. [Cancer Res 2008;68(19):7975–84]

Each of the roughly 100 types of human cancer is caused by the activation of proto-oncogenes and the loss of tumor suppressor genes. Although cancer genomes are complex, some clear mutational patterns are apparent. Several cancer genes are observed very frequently in some types of cancer, but rarely found in other types. Other cancer genes are much more widespread across many types of cancer. Analysis of many cancer genomes has shown that nearly 150 genes are recurrently altered by mutations to provide tumor cells a selective growth advantage. These studies have shown that there are many potential combinations of cancer genes that can cooperatively allow the growth of different types of neoplasia.

The previous chapters have described what cancer genes are and how they are acquired. But what do cancer genes do? How does the inactivation of tumor suppressor genes and activation of proto-oncogenes cause proliferating cells to evolve into cancers? The answer to this question has been revealed by the functional analysis of the proteins encoded by cancer genes and their wild type counterparts.