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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.

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.

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.

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 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.

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.

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]

The phosphatidylinositol 3-kinase-like kinases (PIKKs) ATM and ATR activate a complex signaling network in response to diverse forms of DNA damage. Initial characterization of these signaling molecules focused on the individual role that each plays in response to specific types of DNA lesions. Recently, a more integrated view of the DNA-damage signaling network has emerged. ATM- and ATR-activated signaling pathways once appeared parallel, but new findings suggest that this cellular circuitry is highly interconnected. Communication between ATM and ATR enables the cell to respond to DNA strand breaks and inhibition of DNA synthesis with coordinated, highly modulated outputs. In this article, we focus on several new developments that give insight into the integrated processing of diverse signals that arise during the damage and replication of DNA.

The energy that sustains cancer cells is derived preferentially from glycolysis. This metabolic change, the Warburg effect, was one of the first alterations in cancer cells recognized as conferring a survival advantage. Here, we show that p53, one of the most frequently mutated genes in cancers, modulates the balance between the utilization of respiratory and glycolytic pathways. We identify Synthesis of Cytochrome c Oxidase 2 (SCO2) as the downstream mediator of this effect in mice and human cancer cell lines. SCO2 is critical for regulating the cytochrome c oxidase (COX) complex, the major site of oxygen utilization in the eukaryotic cell. Disruption of theSCO2gene in human cancer cells with wild-type p53 recapitulated the metabolic switch toward glycolysis that is exhibited by p53-deficient cells. That SCO2 couples p53 to mitochondrial respiration provides a possible explanation for the Warburg effect and offers new clues as to how p53 might affect aging and metabolism.

The low level of oxygenation within tumors is a major cause of radiation treatment failures. We theorized that anaerobic bacteria that can selectively destroy the hypoxic regions of tumors would enhance the effects of radiation. To test this hypothesis, we used spores of Clostridium novyi-NT to treat transplanted tumors in mice. The bacteria were found to markedly improve the efficacy of radiotherapy in several of the mouse models tested. Enhancement was noted with external beam radiation derived from a Cs-137 source, systemic radioimmunotherapy with an I-131-conjugated monoclonal antibody, and a previously undescribed form of experimental brachytherapy using plaques loaded with I-125 seeds. C. novyi-NT spores added little toxicity to the radiotherapeutic regimens, and the combination resulted in long-term remissions in a significant fraction of animals.

One of the most productive areas of biologic research has been the utilization of model organisms for the systematic study of gene function. Although the experimental manipulation of these model genetic systems has provided important insights into the function of homologous genes in humans, such studies are necessarily limited by the need to extrapolate among divergent species and cell types. Researchers have now begun to apply the technology of gene targeting to human cell lines. Recently, studies of human cell knockouts have yielded important new information about how the cell cycle is regulated and how this regulation can go awry in cancer cells. The targeting of human genes promises to be a powerful tool in the characterization of the molecular pathways relevant to cancer.

Successful cancer therapy requires the selective killing of cancer cells. Many molecular components of the pathways that lead to cell death have recently been identified and a number of these, including p53, Apaf-1, and members of the inhibitor of apoptosis protein and Bcl-2 gene families, have been found to be altered or disregulated in many cancers. These recent advances and the ongoing elucidation of how these pathways work is providing clues as to how therapeutically resistant cancers might be attacked.

The human DNA damage responses are modulated by both nonessential and essential pathways. The extensively studied ATM kinase and p53 are examples of the former. While loss-of-function mutations in genes that encode ATM and p53 cause marked predispositions to cancer, the loss of these proteins does not appear to impact basic cell growth and proliferation. In contrast, the checkpoint kinase Chk1 and its upstream activator ATR are essential.1-4 What do these proteins do in undamaged cells?

We have isolated a full-length mouse cDNA encoding a lysine-rich protein of 1,131 amino acids with a calculated molecular mass of 126 kDa. The protein binds in a sequence-unspecific manner to DNA, is localized exclusively in the nucleus, and contains a putative ATP binding site and a stretch of 80 amino acids with homology to the carboxy terminus of prokaryotic DNA ligases. On the basis of the following facts, we conclude that the isolated cDNA encodes the 140-kDa subunit of mouse replication factor C (mRFC140). (i) The sequence around the ATP binding site shows significant homology to three small subunits of human replication factor C. (ii) Polyclonal antibodies raised against the protein encoded by this cDNA cross-react with the 140-kDa subunit of purified human replication factor C (hRFC140) and recognize in mouse cell extracts an authentic protein with an apparent molecular mass of 130 kDa. (iii) Sequence comparison with a human cDNA isolated by using tryptic peptide sequence information from purified hRFC140 revealed 83% identity of the encoded proteins. The mRFC140 gene is ubiquitously expressed, and two mRNAs approximately 5.0 and 4.5 kb long have been detected. The gene was mapped by in situ hybridization to mouse chromosome 5, and its human homolog was mapped to chromosome 4 (p13-p14).

Nuclear factor erythroid-2 related factor 2 (Nrf2), a redox-sensitive transcription factor, regulates the expression of antioxidant enzymes and several anti-apoptotic proteins, which confer cytoprotection against oxidative stress and apoptosis. Constitutive activation of Nrf2 in lung cancer cells promotes tumorigenicity and contributes to chemoresistance by upregulation of glutathione, thioredoxin, and the drug efflux pathways involved in detoxification of electrophiles and broad spectrum of drugs. In this study, we show that RNAi-mediated lowering of Nrf2 levels in non-small-cell lung cancer (NSCLC) cell lines (A549 and H460) led to a dramatic increase in endogenous reactive oxygen species (ROS) levels. Similarly, γ-irradiation-induced formation of protein carbonyls were significantly higher in Nrf2-depleted lung cancer cells, suggesting increased lethality of ionizing radiation in the absence of Nrf2. Radiation-induced protein oxidation in Nrf2shRNA cells correlated with reduced survival as measured by clonogenic assay. Radiation-induced cell death was abrogated by pretreatment with antioxidants such as N-acetyl-L-cysteine, glutathione, and vitamin-E, highlighting the importance of antioxidants in conferring protection against radiation injury. Using genetically-modified gain and loss of function models of Nrf2, in mouse embryonic fibroblasts, we establish that constitutive activation of Nrf2 protects against ionizing radiation toxicity and confers radioresistance. Thus, targeting Nrf2 activity in radioresistant tumors could be a promising strategy to circumvent radioresistance. Antioxid. Redox Signal. 13, 1627–1637.

Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that directly transactivates genes important for the growth and metabolism of solid tumors. HIF-1α is overexpressed in cancer, and its level of expression is correlated with patient mortality. Increased synthesis or stability of HIF-1α can be induced by hypoxia-dependent or hypoxia-independent factors. Thus, HIF-1α is expressed in both nonhypoxic and hypoxic cancer cells. The role of HIF-1α in nonhypoxia-mediated cancer cell proliferation remains speculative. We have disrupted HIF-1α by targeted homologous recombination in HCT116 and RKO human colon cancer cells. Loss of HIF-1α significantly reduced nonhypoxia-mediated cell proliferation in vitro and in vivo. Paradoxically, loss of HIF-1α expression did not grossly affect the hypoxic compartments within tumor xenografts in vivo, although HIF-1α promoted cell proliferation and survival under hypoxia in vitro. To further test the role of HIF-1α within tumor compartments, we generated cells with combined disruptions of both HIF-1α and vascular endothelial growth factor (VEGF). In all xenografts, disruption of VEGF led to marked expansion of the hypoxic compartments and growth delay. Nonetheless, the presence or absence of HIF-1α did not grossly affect these expanded hypoxic compartments. These data provide compelling evidence that, in a subset of colon cancers, (a) HIF-1α is a positive factor for nonhypoxia-mediated cell proliferation in vitro and in vivo and (b) HIF-1α is a positive factor for cell proliferation and survival under hypoxic conditions in vitro, but does not grossly contribute to the tumor hypoxic compartments in vivo. (Cancer Res 2006; 66(3): 1684-93)

Replication factor C (RFC) is a multisubunit, DNA polymerase accessory protein required for the coordinated synthesis of both DNA strands during simian virus 40 DNA replication in vitro. Previous studies have shown that RFC is a DNA-dependent ATPase that binds in a structure-specific manner to the 3' end of a primer hybridized to a template DNA, an activity thought intrinsic to the 140-kDa component of this multisubunit complex. Here, the isolation and analysis of cDNAs encoding this subunit is described. Analysis of the full-length coding sequence revealed an open reading frame of 3.4 kb, encoding an 1148-amino acid protein with a predicted molecular mass of 130 kDa. A putative ATP-binding motif was observed that is similar to a motif in several of the smaller subunits of RFC and in functionally homologous replication factors of bacterial and viral origin. A "DEAD" box is also conserved among these proteins. The predicted protein shows significant identity with a DNA-binding protein of murine origin (B. Luckow, P. Lichter, and G. Schütz, personal communication). Regions of similarity were also seen between the amino acid sequences of the 140-kDa subunit of RFC, poly(ADP-ribose) polymerase, and bacterial DNA ligases--possibly representing a conserved structural feature of these proteins that bind similar DNA substrates.

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., Author Summary 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.