PDF file, 244K, BRCA1 foci demonstrate similar kinetics to Rad51 foci after exposure to ionizing radiation.

e16563 Background: Hyperdiploid cells are a hallmark of ovarian cancers that display chemoresistance and poor survival. Aneuploid hyperdiploid (AHD) cells are key targets, because genomic instability confers an evolvable phenotype protected from deleterious mutations by elevated DNA content. Ploidy scales with cell size, increasing metabolic demand and creating a vulnerability to indicate clinically viable metabolic therapies. Methods: We characterised the ploidy, cell size, cell cycle and carboplatin sensitivity of a panel of ovarian cancer cell lines. Anti-proliferative effects of glycolysis (2-deoxyglucose; 2DG) and glutaminolysis (acivicin) antimetabolites were investigated alone and in combinations with carboplatin. Drug interactions were formally defined with compusyn software. Media containing glucose (3mM) and β-hydroxybutyrate (5mM) concentrations reflecting those of blood during a ketogenic diet (ketone media; KM) were tested in combination with carboplatin and acivicin. Results: AHD cell lines were more sensitive to 2DG than diploid A2780 cells. HEY1 and IGROV1 proliferated significantly less in KM than complete media, whereas diploid A2780 cells were unchanged. KM and 2DG in combination with carboplatin increased chemoresistance. However, the interaction between carboplatin and acivicin is synergistic for A2780, and especially for SKOV3. Furthermore, KM increased the overall efficacy, but not potency, of acivicin in A2780 and SKOV3. Conclusions: AHD cells are larger and more sensitive to therapies targeting metabolism than diploid cancer cells, suggesting DNA content and cell size could be biomarkers for these therapies. The simplicity of translating a dietary treatment makes the effect of KM on proliferation in AHD cell lines HEY1 and IGROV1 an intriguing result. SKOV3 had a very synergistic interaction with acivicin and carboplatin, suggesting a corollary sensitivity to glutaminolysis blockade in cells resistant to glucose deprivation. Overall, diploid A2780 cells were less amenable to metabolic therapies, indicating ploidy as a potential biomarker. Ploidy, 2DG IC50, and sensitivity (+) or resistance (-) to KM. Ploidy 2DG IC50 KM A2780 Diploid 19.56mM - HEY1 AHD 1.014mM + IGROV1 AHD 1.811mM + SKOV3 AHD 4.904mM -

INTRODUCTION: Glioblastoma (GBM) remains the most frequent and lethal of all adult brain tumours. Recurrence after therapy particularly contributes to poor outcome. Cancer stem cells (CSC) have been identified as a cellular source that is more resistant to anti-cancer therapy and capable of initiating new tumour growth. We previously found that isolating slow proliferating cells from GBM enriches for a population with CSC properties. METHOD: Using primary patient-derived GBM cells, label-retention assays and flow cytometry, we studied invasion, therapy resistance and stemness of slow-dividing GBM cells in vitro and in vivo. RESULTS: Here, we show that these slow-dividing CSC are more invasive and more resistant to chemotherapy than the rest of the tumour population. Surprisingly, slow-proliferating cells are initially more sensitive to radiation damage. We find a significant overlap between the slow-dividing compartment and expression of the transcription factor ZEB1, which we have recently identified as a master regulator of stemness and chemoresistance in GBM. Consequently, ZEB1+ cells also exhibit greater radiosensitivity. Slow proliferating, ZEB1+ cells accumulate genomic aberrations correlated with retention in the G2/M phase of the cell cycle, rendering these cells more sensitive to radiation damage. However, from this specific subpopulation, a fraction of cells that survive irradiation rebound with a proliferative burst that may contribute to recurrence of more aggressive tumours. CONCLUSION: This disparate effect of radiation on CSC points to a previously underappreciated heterogeneity within the CSC compartment and may open up new avenues of studying and targeting specific CSC sub-populations.

Tumor evolution presents a formidable obstacle that currently prevents the development of truly curative treatments for cancer. In this perspective, we advocate for the hypothesis that tumor cells with significantly elevated genomic content (polyploid tumor cells) facilitate rapid tumor evolution and the acquisition of therapy resistance in multiple incurable cancers. We appeal to studies conducted in yeast, cancer models, and cancer patients, which all converge on the hypothesis that polyploidy enables large phenotypic leaps, providing access to many different therapy-resistant phenotypes. We develop a flow-cytometry based method for quantifying the prevalence of polyploid tumor cells, and show the frequency of these cells in patient tumors may be higher than is generally appreciated. We then present recent studies identifying promising new therapeutic strategies that could be used to specifically target polyploid tumor cells in cancer patients. We argue that these therapeutic approaches should be incorporated into new treatment strategies aimed at blocking tumor evolution by killing the highly evolvable, therapy-resistant polyploid cell subpopulations, thus helping to maintain patient tumors in a drug sensitive state.

Here we report the identification of a proliferative, viable, and hyperdiploid tumor cell subpopulation present within Glioblastoma (GB) patient tumors. Using xenograft tumor models, we demonstrate that hyperdiploid cell populations are maintained in xenograft tumors and that clonally expanded hyperdiploid cells support tumor formation and progression in vivo. In some patient tumorsphere lines, hyperdiploidy is maintained during long-term culture and in vivo within xenograft tumor models, suggesting that hyperdiploidy can be a stable cell state. In other patient lines hyperdiploid cells display genetic drift in vitro and in vivo, suggesting that in these patients hyperdiploidy is a transient cell state that generates novel phenotypes, potentially facilitating rapid tumor evolution. We show that the hyperdiploid cells are resistant to conventional therapy, in part due to infrequent cell division due to a delay in the G₀/G₁ phase of the cell cycle. Hyperdiploid tumor cells are significantly larger and more metabolically active than euploid cancer cells, and this correlates to an increased sensitivity to the effects of glycolysis inhibition. Together these data identify GB hyperdiploid tumor cells as a potentially important subpopulation of cells that are well positioned to contribute to tumor evolution and disease recurrence in adult brain cancer patients, and suggest tumor metabolism as a promising point of therapeutic intervention against this subpopulation.

Glioblastoma, the most frequent primary malignancy of the central nervous system, is almost universally fatal despite aggressive therapies, such as surgical resection, adjuvant radiation and chemotherapy, which remain largely palliative. With increasing evidence showing that glioblastoma cancer stem cells play an important role in tumor escape from conventional therapies and disease recurrence, the targeting of cancer stem cells with different therapeutic strategies provides new avenues of research and confidence for better outcomes. We have previously shown that isolating slow-dividing cells from glioblastoma enriches for a population with cancer stem cell properties. Here, we demonstrate that these slow-dividing cancer stem cells are more invasive and more tolerant to chemotherapy than the rest of the tumor population. Surprisingly, slow-proliferating cells are initially more sensitive to radiation damage. We find a significant overlap between the slow-proliferating compartment and expression of the transcription factor ZEB1, which we have recently identified as a master regulator of stemness and chemoresistance in glioblastoma. Consequently, ZEB1-positive cells also exhibit greater radiosensitivity. Slow proliferating, ZEB1-positive cells accumulate genomic aberrations correlated with retention in the G2/M phase of the cell cycle, rendering these cells more sensitive to radiation damage. However, from this specific subpopulation, a fraction of cells that survive irradiation rebound with a proliferative burst that may contribute to recurrence of more aggressive tumors. This distinct effect of radiation on cancer stem cells points to a previously underappreciated heterogeneity within the cancer stem cell compartment and may open up new avenues of studying and targeting specific cancer stem cell sub-populations.

Genetic and biochemical studies have revealed that the diversity of cell types and developmental patterns evident within the animal kingdom is generated by a handful of conserved, core modules. Core biological modules must be robust, able to maintain functionality despite perturbations, and yet sufficiently adaptable for random mutations to generate phenotypic variation during evolution. Understanding how robust, adaptable modules have influenced the evolution of eukaryotes will inform both evolutionary and synthetic biology. One such system is the MAP kinase module, which consists of a 3-tiered kinase circuit configuration that has been evolutionarily conserved from yeast to man. MAP kinase signal transduction pathways are used across eukaryotic phyla to drive biological functions that are crucial for life. Here we ask the fundamental question, why do MAPK modules follow a conserved 3-tiered topology rather than some other number? Using computational simulations, we identify a fundamental 2-tiered circuit topology that can be readily reconfigured by feedback loops and scaffolds to generate diverse signal outputs. When this 2-kinase circuit is connected to proximal input kinases, a 3-tiered modular configuration is created that is both robust and adaptable, providing a biological circuit that can regulate multiple phenotypes and maintain functionality in an uncertain world. We propose that the 3-tiered signal transduction module has been conserved through positive selection, because it facilitated the generation of phenotypic variation during eukaryotic evolution.

Significant endeavor has been applied to identify functional therapeutic targets in glioblastoma (GBM) to halt the growth of this aggressive cancer. We show that the receptor tyrosine kinase EphA3 is frequently overexpressed in GBM and, in particular, in the most aggressive mesenchymal subtype. Importantly, EphA3 is highly expressed on the tumor-initiating cell population in glioma and appears critically involved in maintaining tumor cells in a less differentiated state by modulating mitogen-activated protein kinase signaling. EphA3 knockdown or depletion of EphA3-positive tumor cells reduced tumorigenic potential to a degree comparable to treatment with a therapeutic radiolabelled EphA3-specific monoclonal antibody. These results identify EphA3 as a functional, targetable receptor in GBM.

AbstractGlioblastoma multiforme (GBM) is the most common form of brain tumor with a poor prognosis and resistance to radiotherapy. Recent evidence suggests that glioma-initiating cells play a central role in radioresistance through DNA damage checkpoint activation and enhanced DNA repair. To investigate this in more detail, we compared the DNA damage response in nontumor forming neural progenitor cells (NPC) and glioma-initiating cells isolated from GBM patient specimens. As observed for GBM tumors, initial characterization showed that glioma-initiating cells have long-term self-renewal capacity. They express markers identical to NPCs and have the ability to form tumors in an animal model. In addition, these cells are radioresistant to varying degrees, which could not be explained by enhanced nonhomologous end joining (NHEJ). Indeed, NHEJ in glioma-initiating cells was equivalent, or in some cases reduced, as compared with NPCs. However, there was evidence for more efficient homologous recombination repair in glioma-initiating cells. We did not observe a prolonged cell cycle nor enhanced basal activation of checkpoint proteins as reported previously. Rather, cell-cycle defects in the G1–S and S-phase checkpoints were observed by determining entry into S-phase and radioresistant DNA synthesis following irradiation. These data suggest that homologous recombination and cell-cycle checkpoint abnormalities may contribute to the radioresistance of glioma-initiating cells and that both processes may be suitable targets for therapy. Mol Cancer Ther; 11(9); 1863–72. ©2012 AACR.

Evolution is often characterized as a process involving incremental genetic changes that are slowly discovered and fixed in a population through genetic drift and selection. However, a growing body of evidence is finding that changes in the environment frequently induce adaptations that are much too rapid to occur by an incremental genetic search process. Rapid evolution is hypothesized to be facilitated by mutations present within the population that are silent or “cryptic” within the first environment but are co-opted or “exapted” to the new environment, providing a selective advantage once revealed. Although cryptic mutations have recently been shown to facilitate evolution in RNA enzymes, their role in the evolution of complex phenotypes has not been proven. In support of this wider role, this paper describes an unambiguous relationship between cryptic genetic variation and complex phenotypic responses within the immune system. By reviewing the biology of the adaptive immune system through the lens of evolution, we show that T cell adaptive immunity constitutes an exemplary model system where cryptic alleles drive rapid adaptation of complex traits. In naive T cells, normally cryptic differences in T cell receptor reveal diversity in activation responses when the cellular population is presented with a novel environment during infection. We summarize how the adaptive immune response presents a well studied and appropriate experimental system that can be used to confirm and expand upon theoretical evolutionary models describing how seemingly small and innocuous mutations can drive rapid cellular evolution.

Abstract Purpose: Eph receptors constitute the largest sub-family of receptor tyrosine kinases and interact with membrane-bound ligands termed ephrins. Eph and ephrins have many vital functions including cell adhesion, migration and axon guidance. Eph and ephrins have been found to be aberrantly expressed in many malignancies including brain tumor. The purpose of this study was to investigate EphA3 receptor function in the most common and aggressive form of brain tumor, Glioblastoma (GBM). Methods: Gene expression was investigated by Q-PCR, IHC and flow cytometry in high grade glioma (HGG) surgical specimens and primary derived serum free cell cultures. Targeted reduction of Eph expression was performed using both a constitutive and inducible shRNA system. Murine in-vivo studies were performed using both subcutaneous and orthotopic ‘intracranial’ xenografts in immuno-compromised animals. Signaling pathways were assessed by western blotting. Results: To establish whether the receptor tyrosine kinase EphA3 was over expressed in HGG we assessed 12 normal human brain specimens, 56 HGG specimens and 26 HGG primary cultures. EphA3 mRNA expression was negligible in normal brain while 30% of clinical specimens and 46% of primary cultured tumor cells expressed EphA3. EphA3 protein was also detected in HGG clinical specimens using IHC. To further investigate EphA3 function the receptor was down regulated using shRNA in an EphA3+ GBM neurosphere cell line. Constitutive and inducible down regulation of the EphA3 receptor resulted in initiation of neuronal and glial cell differentiation following activation of the ERK/MAPK pathway. A reduction in stem/progenitor cell proliferation was also observed following EphA3 knockdown by shRNA (46%) or by alternately inhibiting EphA3 function using soluble ephrin A5-Fc (33%). CFSE division tracking identified slower cell division in populations in which EphA3 signaling was attenuated. In-vivo studies were performed using a NOD/SCID mouse subcutaneous and intracranial xenograft model. Results highlighted a marked reduction in tumor formation in the EphA3 knockdown as opposed to control tumors. Subcutaneous control tumors formed with a median survival of 66 days while EphA3 knockdown animals survived beyond 100 days (p<0.05). Similar to the subcutaneous xenograft model a marked lack of intracranial tumor formation was observed when EphA3 was neutralized. Control mice formed large well vascularized invasive tumors with a median survival of 62 days. All EphA3 knockdown animals were free of tumor following autopsy at 145 days when the experiment was terminated (p<0.05). Importantly a mutant EphA3 rescue of the knockdown culture returned the tumorigenic potential of these cells. Conclusions: We propose EphA3, in part, regulates cancer stem cell self renewal and cell division rate in GBM and could prove a potential therapeutic target and marker of brain tumor initiating stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1197. doi:10.1158/1538-7445.AM2011-1197

Individual tumour cells display diverse functional behaviours in terms of proliferation rate, cell-cell interactions, metastatic potential and sensitivity to therapy. Moreover, sequencing studies have demonstrated surprising levels of genetic diversity between individual patient tumours of the same type. Tumour heterogeneity presents a significant therapeutic challenge as diverse cell types within a tumour can respond differently to therapies, and inter-patient heterogeneity may prevent the development of general treatments for cancer. One strategy that may help overcome tumour heterogeneity is the identification of tumour sub-populations that drive specific disease pathologies for the development of therapies targeting these clinically relevant sub-populations. Here, we have identified a dye-retaining brain tumour population that displays all the hallmarks of a tumour-initiating sub-population. Using a limiting dilution transplantation assay in immunocompromised mice, label-retaining brain tumour cells display elevated tumour-initiation properties relative to the bulk population. Importantly, tumours generated from these label-retaining cells exhibit all the pathological features of the primary disease. Together, these findings confirm dye-retaining brain tumour cells exhibit tumour-initiation ability and are therefore viable targets for the development of therapeutics targeting this sub-population.

Unless diagnosed early, many adult cancers remain incurable diseases. This is despite an intense global research effort to develop effective anticancer therapies, calling into question the use of rational drug design strategies in targeting complex disease states such as cancer. A fundamental challenge facing researchers and clinicians is that cancers are inherently robust biological systems, able to survive, adapt and proliferate despite the perturbations resulting from anticancer drugs. It is essential that the mechanisms underlying tumor robustness be formally studied and characterized, as without a thorough understanding of the principles of tumor robustness, strategies to overcome therapy resistance are unlikely to be found. Degeneracy describes the ability of structurally distinct system components (e.g. proteins, pathways, cells, organisms) to be conditionally interchangeable in their contribution to system traits and it has been broadly implicated in the robustness and evolvability of complex biological systems. Here we focus on one of the most important mechanisms underpinning tumor robustness and degeneracy, the cellular heterogeneity that is the hallmark of most solid tumors. Based on a combination of computational, experimental and clinical studies we argue that stochastic noise is an underlying cause of tumor heterogeneity and particularly degeneracy. Drawing from a number of recent data sets, we propose an integrative model for the evolution of therapy resistance, and discuss recent computational studies that propose new therapeutic strategies aimed at defeating the adaptable cancer phenotype.

For some years laboratories working with ancient DNA have been optimising extraction methods using either undamaged modern DNA or authentic ancient DNA. This approach is unsatisfactory for a number of reasons, chief being the inherent variability from sample to sample. In addition, quantitative comparison of methods is generally impossible using typically small samples of ancient DNA, as well as being ethically questionable. We have now perfected a method whereby we can oxidatively damage the plasmid pUC19 using copper sulfate, ascorbic acid and hydrogen peroxide to create artificially damaged DNA that mimics the behaviour of ancient DNA. We have used this damaged plasmid to assay our extraction methods to quantitatively monitor the yield and degree of damage induced during the extraction, purification, and storage of DNA. We suggest that the damaged plasmid can be used as a monitoring standard within the one laboratory, as we have done, and more importantly to compare yields and efficiencies between different laboratories.