Genetic Instability Of Cancer Cells Research Articles

Harnessing Light for G-Quadruplex Modulation: Dual Isomeric Effects of an Ortho-Fluoroazobenzene Derivative.

G-quadruplexes (G4s) are important therapeutic and photopharmacological targets in cancer research. Small-molecule ligands targeting G4s offer a promising strategy to block DNA transactions and induce genetic instability in cancer cells. While numerous G4-ligands have been reported, relatively few examples exist of compounds whose G4-interactive binding properties can be modulated using light. Herein, we report the photophysical characterization of a novel ortho-fluoroazobenzene derivative, Py-Azo4F-3N, that undergoes reversible two-way isomerization upon visible light exposure. Using a combination of biophysical techniques, including affinity and selectivity assays, structural and computational analysis, and cytotoxicity experiments in cancer cell lines, we carefully characterized the G4-interactive binding properties of both isomers. We identify the trans isomer as the most promising form of interacting and stabilizing G4s, enhancing their ablation capability in cancer cells. Our research highlights the importance of light-responsive molecules in achieving precise control over G4 structures, demonstrating their potential in innovative anticancer strategies.

Impact of R-loops on oncogene-induced replication stress in cancer cells.

Replication stress is an alteration in the progression of replication forks caused by a variety of events of endogenous or exogenous origin. In precancerous lesions, this stress is exacerbated by the deregulation of oncogenic pathways, which notably disrupts the coordination between replication and transcription, and leads to genetic instability and cancer development. It is now well established that transcription can interfere with genome replication in different ways, such as head-on collisions between polymerases, accumulation of positive DNA supercoils or formation of R-loops. These structures form during transcription when nascent RNA reanneals with DNA behind the RNA polymerase, forming a stable DNA:RNA hybrid. In this review, we discuss how these different cotranscriptional processes disrupt the progression of replication forks and how they contribute to genetic instability in cancer cells.

Interaction Among Noncoding RNAs, DNA Damage Reactions, and Genomic Instability in the Hypoxic Tumor: Is it Therapeutically Exploitable Practice?

Hypoxia is a classical function of the tumor's microenvironment with a substantial effect on the development and therapeutic response of cancer. When put in hypoxic environments, cells undergo several biological reactions, including activation of signaling pathways that control proliferation, angiogenesis, and death. These pathways have been adapted by cancer cells to allow tumors to survive and even develop in hypoxic conditions, and poor prognosis is associated with tumor hypoxia. The most relevant transcriptional regulator in response to hypoxia, Hypoxia-inducible factor-1 alpha (HIF-1α), has been shown to modulate hypoxic gene expression and signaling transduction networks significantly. The significance of non-coding RNAs in hypoxic tumor regions has been revealed in an increasing number of studies over the past few decades. In regulating hypoxic gene expression, these hypoxia-responsive ncRNAs play pivotal roles. Hypoxia, a general characteristic of the tumor's microenvironment, significantly affects the expression of genes and is closely associated with the development of cancer. Indeed, the number of known hypoxia-associated lncRNAs has increased dramatically, demonstrating the growing role of lncRNAs in cascades and responses to hypoxia signaling. Decades of research have helped us create an image of the shift in hypoxic cancer cells' DNA repair capabilities. Emerging evidence suggests that hypoxia can trigger genetic instability in cancer cells because of microenvironmental tumor stress. Researchers have found that critical genes' expression is coordinately repressed by hypoxia within the DNA damage and repair pathways. In this study, we include an update of current knowledge on the presentation, participation, and potential clinical effect of ncRNAs in tumor hypoxia, DNA damage reactions, and genomic instability, with a specific emphasis on their unusual cascade of molecular regulation and malignant progression induced by hypoxia.

Abstract 346: Synergistic antiproliferative activity of novel RAD51 inhibitor JKYN-1 and its mesylate salt with standard-of-care cancer drugs

Abstract The inherent genetic instability of cancer cells and the dependence of many tumor types on oncogenic drivers contribute selectivity of anticancer agents against tumor cells. That selectivity is limited, and toxicity to normal cells remains a major limitation to the success of chemotherapy. To increase selectivity by exploiting cancer cell genetic instability, we demonstrated that the small molecule IBR2 (an inhibitor of the DNA repair protein RAD51) enhanced cytotoxicity of numerous anticancer drugs including agents that do not directly target DNA (J Pharmacol Expt Ther, 364: 46-54, 2018. doi.org/10.1124/jpet.117.241661). We demonstrated the ability of IBR2 and a derivative [IBR120, (R)-3-(2-(benzylsulfonyl)isoindolin-1-yl)-1h-indole] to synergistically inhibit proliferation of a wider range of cancer cell lines in combination with a broad range of anticancer drugs (Proc. Amer. Assoc. Cancer Res., 60: Abst. 3057, 2019). To improve the activity and potentially increase selectivity for inhibiting RAD51, modifications were made to the structure of IBR120 using a virtual drug-protein docking program, yielding the compound JKYN-1. JKYN-1 inhibits proliferation of cancer cell lines approximately 5 times more strongly than IBR120. Given the potential importance of combining JKYN-1 with targeted anticancer drugs to increase therapeutic index, and the synergy previously observed between IBR120 and agents targeted against specific tumor types, JKYN-1 was tested in combination with targeted agents against a panel of tumor cell lines. Four- to five-day drug exposures were conducted in 96-well plates. Relative cell density determined using vital stains (alamarBlue©, neutral red) was reported as a percent of the fluorescence/absorbance of control cultures. Cell lines were representative of tumors from breast (MCF-7), prostate (DU145, LNCaP), stomach (N87), pancreas (PANC-1, Capan-1, Capan-2) and lung (A549b, H1650). The chemotherapy agents included inhibitors of epidermal growth factor receptor (osimertinib, afatinib), other tyrosine kinases (regorafenib, imatinib), sex steroid receptors (4-OH-tamoxifen, enzalutamide), and microtubule function (docetaxel). To improve solubility, a methylsulfonate salt of JKYN-1 was used for most experiments. JKYN-1-mesylate decreased the concentration of drugs that inhibited proliferation by 50% (IC50) by up to 90%, depending on the drug and cell line, indicating synergy between the agents. There were some combinations in which additivity but no synergy was observed, indicating selectivity for this interaction. Individual combinations will be presented. The ability of JKYN-1 to enhance antiproliferative activity of a wide variety of anticancer agents, and its potential selectivity for cancer cells, make possible the future use of RAD51 inhibitors as systemic therapy potentiators to improve clinical outcomes. Citation Format: Peter J. Ferguson, Mark D. Vincent, Yousef Najajreh, Brian Shilton, Stephen Ritter, Rima Al-awar, Richard Marcellus, Mohammed Mohammed, Methvin Isaac, James Koropatnick. Synergistic antiproliferative activity of novel RAD51 inhibitor JKYN-1 and its mesylate salt with standard-of-care cancer drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 346.

KV11.1 Channel Activity Control Reactive Oxygen Species (ROS) Homeostasis in Breast Cancer Cells

Generation and control of reactive oxygen species (redox system) is essential for maintaining cellular homeostasis. Increased oxidative stress can play a critical role in a variety of pathological condition including cancer. It is known that cancer cells present higher level of DNA oxidation which is dependent of downregulation of antioxidant enzymes such as the Nuclear factor erythroid 2-related factor 2 (NRF2). This phenomenon underlies the characteristic genetic instability of cancer cells and is a feature of neoplastic lesions. Therefore, activation of antioxidant enzymes received attention as a potential anticancer strategy. However, the biochemical cascades controlling the redox system in cancer cell survival or death is not clearly understood. We discovered that activity of the Kv11.1 channel (hERG1) arrests breast cancer proliferation by activating a senescent-like phenotype that coincides with increased ROS formation. We found that stimulation of Kv11.1 channel with selective activator molecules (NS1643 or PD118057) produces a significant alteration of mitochondrial function which in turn increases ROS production. Interestingly, cancer cells respond to the Kv11.1-dependent increase of ROS by activation of the NRF2 transcription factor leading to an increased production of antioxidant proteins. Therefore, the NRF2 protein promotes cell survival. Interestingly, the combination of the Kv11.1 activator NS1643 with suppression of NRF2 function produced a significant increase of ROS which in turn leads to cell death. Our data demonstrate that activity of the Kv11.1 channel control mitochondria function and ROS formation in breast cancer cells. Remarkably, these cells activate transcription of antioxidant enzymes via NRF2 which acts as survival mechanism.

Abstract 3057: Synergistic antiproliferative activity between anticancer drugs and RAD51 inhibitors IBR2 and IBR120

Abstract The inherent genetic instability of cancer cells and the dependence of many tumor types on oncogenic drivers contribute selectivity of anticancer agents against tumor cells. However, that selectivity is limited, and toxicity to normal cells remains a major limitation to the success of chemotherapy. Aiming to increase selectivity by exploiting cancer cell genetic instability, we demonstrated that the small molecule IBR2 (an inhibitor of the DNA repair protein RAD51) enhanced cytotoxicity of numerous anticancer drugs, including agents that do not directly target DNA (J Pharmacol Expt Ther, 364: 46-54, 2018). Given the potential importance of combining IBR2 with anticancer treatment to increase therapeutic index, IBR2 was tested in combination with other untested agents against a panel of tumor cell lines. In addition, studies were expanded to include IBR120 [(R)-3-(2-(benzylsulfonyl)isoindolin-1-yl)-1h-indole], a new small molecule RAD51 inhibitor that is reportedly more selective against cancer cell lines (Euro J Med Chem 96: 196-208, 2015). Four-day drug exposures were conducted in 96-well plates. Relative cell density, determined using vital stains (alamarBlue©, neutral red), was reported as a percent of the fluorescence/absorbance of control cultures. Cell lines were representative of tumors from breast (MCF-7, SK-BR-3), prostate (LNCaP, DU145), colon (HT-29), stomach (N87), skin (SK-MEL-5), and lung (A549b, H1650, H1975), including osimertinib-resistant derivatives of the non-small-cell lung cancer (NSCLC) cell lines. The chemotherapy agents included inhibitors of epidermal growth factor receptor (EGFR) family (osimertinib, afatinib, lapatinib), Bcr-Abl (imatinib), B-raf (vemurafenib), multiple tyrosine kinases (regorafenib), sex steroid receptors (4-OH-tamoxifen, enzalutamide), and microtubules (vincristine). Both IBR2 and IBR120 decreased the concentration of drugs that inhibited proliferation by 50% (IC50) by up to 90%, indicating synergy between the agents. However, there were a small number of combinations in which no synergy was observed, indicating selectivity for this interaction. In the NSCLC cell lines, IBR2, IBR120, osimertinib, gefitinib, and afatinib decreased the cellular content of RAD51 by over 80% within 24 h (western blot with antibody 3C10), at concentrations at or lower than their IC50 value. The contribution of this decrease in RAD51 to synergy is being investigated. Studies are ongoing to determine the effect of IBR120 on growth of explants of the NSCLC cell line H1650 in mice, after which the combination of IBR120 and osimertinib will be tested in vivo. The ability of IBR2 and IBR120 to enhance antiproliferative activity of a wide variety of anticancer agents and their potential selectivity for cancer cells make possible their future use as systemic therapy potentiators to improve clinical outcomes. Citation Format: Peter J. Ferguson, Morgan Black, Rene Figueredo, Mark D. Vincent, James Koropatnick. Synergistic antiproliferative activity between anticancer drugs and RAD51 inhibitors IBR2 and IBR120 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3057.

Intrinsic cancer vaccination

Immunotherapy is revolutionizing the treatment of cancer, and the current immunotherapeutics have remarkably improved the outcomes for some cancer patients. However, we still need answers for patients with immunologically cold tumors that do not benefit from the current immunotherapy treatments. Here, we suggest a novel strategy that is based on using a very old and sophisticated system for cancer immunotherapy, namely "intrinsic cancer vaccination", which seeks to awaken our own immune system to activate tumor-specific T cells. To do this, we must take advantage of the genetic instability of cancer cells and the expression of cancer cell neoantigens to trigger immunity against cancer cells. It will be necessary to not only enhance the phagocytosis of cancer cells by antigen presenting cells but also induce immunogenic cancer cell death and the subsequent immunogenic clearance, cross-priming and generation of tumor-specific T cells. This strategy will allow us to avoid using known tumor-specific antigens, ex vivo manipulation or adoptive cell therapy; rather, we will efficiently present cancer cell neoantigens to our immune system and propagate the cancer-immunity cycle. This strategy simply follows the natural cycle of cancer-immunity from its very first step, and therefore could be combined with any other treatment modality to yield enhanced efficacy.

The Role of HSP70 in Cancer and its Exploitation as a Therapeutic Target.

Sustained proliferation and genetic instability of cancer cells are associated with enhanced production of mutated and conformationally unstable proteins. Excessive proteosynthesis along with increased metabolic turnover generates stress conditions that cancer cells must permanently compensate for. Tumor cells thus become dependent on the maintenance of protein homeostasis, which involves protein quality control, folding, transport and stabilization. These tasks are provided by molecular chaperones, predominantly the stress proteins HSP70 and HSP90. Their expression and activity is increased in all malignant tumors, where they associate with their cochaperones to form large multiprotein complexes. HSP70 and HSP90 maintain the malignant phenotype because they facilitate the folding of numerous oncogenic proteins, maintain proliferative potential, and inhibit apoptosis. In this regard, heat-shock proteins represent an important target for cancer therapy because their inactivation results in the simultaneous blockade of multiple signaling pathways. Although several specific HSP90 inhibitors have been developed in the past decade, their antitumor activity as single agents is limited due to the induction of HSP70, which enables cell survival. Inhibitors of HSP70 thus present new possibilities for targeting proteostatic mechanisms in cancer cells. The aim of this article is to summarize information on the structure of HSP70 and its role in maintaining protein homeostasis in normal and cancer cells. The mechanisms of HSP70 inhibition by low-molecular weight compounds and their application in targeted antitumor therapy are also described. Key words: HSP70 - stress proteins - molecular chaperons - cellular stress - tumours - protein folding This work was supported by the project MEYS - NPS I - LO1413. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Accepted: 16. 08. 2018.

Heterogeneity in untreated, stressed and drug-tolerant cells: insights into the evolution of cancer resistance

The evolution of cancer is a Darwinian process, the composition of cancer cell population varying with time, especially after chemotherapy. Such changes occurring in the genetic landscape of tumours are possibly responsible for the chemotherapeutic resistance. Due to the recent advances in single cellular sequencing, the evolution of cancers can be monitored at the single cellular resolution. We reanalysed a previously published dataset composed of single cell RNAseq data, collected before, during and after the establishment of Paclitaxel tolerance, to identify single cell heterogeneity. We used gene expression analysis, gene expression rank correlation analysis, pathway analysis and mutation analysis to identify within group variations of cancer cells after chemotherapy. We identified a decrease in cancer cell within group diversity during the transition to drug-tolerance. Additionally, we observed high mutation rate in stressed single cells, which suggests genetic instability of cancer cells that could ultimately result in the development of drug resistance. Our analysis carries significant implications for developing personalised and efficient therapeutics against cancer.

Abstract PR04: mTOR mutations in cancer

Abstract Genes in the PI3K-Akt-mTOR signaling axis are frequently mutated in cancer but relatively few mutations have been observed in the MTOR gene itself. By mining publicly available cancer sequencing data, we recently reported that MTOR is commonly mutated in cancer, with more than 35 previously uncharacterized pathway activating mutations. Most were recurrent, localized within distinct clusters in the C-terminal half of the protein, and induced activation of mTOR Complex 1 (mTORC1), mTORC2, or both. The mutations did not prevent mTOR inhibition by rapamycin or ATP-competitive inhibitors, although several conferred resistance to nutrient withdrawal. Importantly, cancer cell lines with activating mTOR mutations are especially sensitive to rapamycin both in vitro and in vivo, indicating pathway dependency. In a companion report, we also demonstrated that the tumor of a bladder cancer patient with exquisite sensitivity to a rapamycin analog (rapalog) harbored two activating mTOR mutations, likely driving mTOR pathway dependency. However, after 14 months of rapalog response, the patient displayed progressive disease, indicating that the tumor had acquired resistance to the targeted therapy. Here, we demonstrate two mechanisms by which cancers may display resistance to rapamycin through mTOR mutation. In the first example, we acquired a tumor biopsy from an anaplastic thyroid carcinoma patient before they were enrolled on a rapalog clinical trial. The patient displayed a durable 18 month response to the rapalog before progressing, at which time we acquired a second biopsy. Whole exome sequencing of the biopsies revealed the presence of a loss-of-function mutation in the mTORC1 negative regulator Tuberin (TSC2), likely driving pathway addiction and thus sensitivity to the rapalog. Sequencing of the post-resistance biopsy revealed that the tumor had acquired a mutation in the rapamycin-binding domain of mTOR. Cells made to exogenously express this mutation displayed potent resistance to rapamycin-mediated inhibition of cell proliferation and phosphorylation of the mTORC1 substrate S6K1. Importantly, the cells remained sensitive to an ATP-competitive mTOR inhibitor because the two classes of mTOR inhibitors rely upon completely different mechanisms of action. Thus, we show that as patients acquire resistance to rapalogs, they may still be effectively treated with ATP-competitive mTOR inhibitors. The rapalog-resistant mutation described above is an example of an acquired mutation, although due to the genetic instability of cancer cells, a tumor may harbor pre-existing rapamycin-resistant mutations. To determine the prevalence of such mutations, we again turned to publicly available cancer sequencing data, identifying approximately 30 mutations in the rapamycin-binding domain of mTOR. The majority these mutations did not alter mTOR pathway activation and remained rapamycin sensitive. Importantly, one breast cancer sample harbored a rapamycin-resistant mTOR mutation, although it remained sensitive to an ATP-competitive mTOR inhibitor. This rare event is highly relevant given that rapalogs are currently approved for the treatment of breast cancer. Had this patient been treated with a rapalog, they would have only experienced the side-effects and none of the benefits of treatment. Collectively, we demonstrate that mTOR mutations are common in cancer, many of which confer pathway activation and addiction. Additionally, both acquired and pre-existing mTOR mutations can confer resistance towards rapamycin treatment, although this resistance can be overcome with ATP-competitive inhibitors. This work has important ramifications for the treatment of cancer patients with mTOR inhibitors, by identifying both those patients likely to respond or display resistance to such treatments. This abstract is also being presented as Poster A44. Citation Format: Brian Grabiner, Nikhil Wagle, Eliezer Van Allen, Levi Garraway, Jochen Lorch, David Sabatini. mTOR mutations in cancer. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr PR04.

HIF1-regulated ATRIP expression is required for hypoxia induced ATR activation

The ATR–ATRIP protein kinase complex plays a crucial role in the cellular response to replication stress and DNA damage. Recent studies found that ATR could be activated in response to hypoxia and be involved in hypoxia-induced genetic instability in cancer cells. However, the underlying mechanisms for ATR activation in response to hypoxic stress are still not fully understood. We reported that ATRIP is a direct target of HIF-1. Silencing the expression of HIF-1α in cancer cells by RNA interference abolished hypoxia-induced ATRIP expression. Silencing the expression of ATRIP by RNA interference abolished hypoxia induced ATR activation and CHK1 phosphorylation in cancer cells. Taken together, these data shed novel insights on the mechanism of hypoxia-induced activation of the ATR pathway.

Molecular Pathways: Comparing the Effects of Drugs and T Cells to Effectively Target Oncogenes

Mutant cancer-driving oncogenes are the best therapeutic targets, both with drugs like small-molecule inhibitors (SMI) and adoptive T-cell therapy (ATT), the most effective form of immunotherapy. Cancer cell survival often depends on oncogenes, which implies that they are homogeneously expressed by all cancer cells and are difficult to select against. Mutant oncogene-directed therapy is relatively selective, as it targets preferentially the oncogene-expressing cancer cells. Both SMI and ATT can be highly effective in relevant preclinical models as well as selected clinical situations, and both share the risk of therapy resistance, facilitated by the frequent genetic instability of cancer cells. Recently, both therapies were compared in the same experimental model targeting the same oncogene. It showed that the oncogene-inactivating drug selected resistant clones, leading eventually to tumor relapse, whereas ATT eradicated large established tumors completely. The mode of tumor destruction likely explained the different outcome with only ATT destroying the tumor vasculature. Elucidating the cellular and molecular mechanisms responsible for tumor regression and relapse will define optimal conditions for the clinic. We argue that the ideal conditions of ATT in the experimental cancer model can be translated to individuals with cancer.

Abstract 3939: Hypoxia-induced BRCA1 phosphorylation and degradation

Abstract Hypoxia is a key feature of tumor microenviroment. We have demonstrated that hypoxia downregulates BRCA1 at the transcriptional level through promoter binding by E2F4/p130 complexes and that hypoxia can produce BRCA1 gene silencing via epigenetic modifications. We also demonstrated that downregulation of BRCA1 by hypoxia leads to decreased DNA repair capacity, establishing a mechanism by which hypoxia can drive genetic instability in cancer cells. In addition to transcriptional regulation, BRCA1 has been found to be phosphorylated at S988 in a Chk2-dependent manner by both ionizing radiation and hypoxia exposure. In this study, we have been able to confirm that other residues of BRCA1 are phosphoylated during hypoxia exposure, including S1387, S1423, S1466, and S1524. Intriguingly, the phosphoryation of BRCA1 only happens under severe hypoxic condition, such as 0.01% oxygen, but not under moderate hypoxia (1% oxygen condition). Furthermore, the BRCA1 phosphoylation events induced by hypoxia can be attenuated by an ATM inhibitor, KU55933, indicating that ATM is playing a role in hypoxia-induced BRCA1 phosphorylation at multiple sites. We also observed that the decrease in BRCA1 protein levels in hypoxia is partially blocked by MG132, a proteasome inhibitor. Consistent with this, by transient transfection of exogenous BRCA1, we found that hypoxia induces BRCA1 unbiquitination in MCF-7 cells. Together, our data suggest that hypoxia-induced downregulation of BRCA1 is in part mediated through the ubiqutin-proteasome pathway. Our findings indicate a new mechanism of hypoxia induced regulation of BRCA1 by modulating its stability, suggesting that hypoxia-induced downregulation of BRCA1 is through multiple mechanisms. Future studies will investigate the mechanism of hypoxia-induced BRCA1 phophorylation and the relation between hypoxia-induced BRCA1 phosphorylation and BRCA1 degradation. To understand more about regulation of BRCA1 by hypoxia could lead to new therapies to target hypoxic tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3939. doi:1538-7445.AM2012-3939

Oncogene-Targeting T Cells Reject Large Tumors while Oncogene Inactivation Selects Escape Variants in Mouse Models of Cancer

The genetic instability of cancer cells frequently causes drug resistance. We established mouse cancer models, which allowed targeting of an oncogene by drug-mediated inactivation or monospecific CD8(+) effector T (T(E)) cells. Drug treatment of genetically unstable large tumors was effective but selected resistant clones in the long term. In contrast, T(E) cells completely rejected large tumors (≥500mm(3)), if the target antigen was cancer-driving and expressed in sufficient amounts. Although drug-mediated oncogene inactivation selectively killed the cancer cells and left the tumor vasculature intact, which likely facilitated survival and growth of resistant clones, T(E) cell treatment led to blood vessel destruction and probably "bystander" elimination of escape variants, which did not require antigen cross-presentation by stromal cells.

When Genetic Instability Is a Good Thing

Many years of cell-cycle research have been devoted to understanding the mechanisms that replicate and repair the genome so that cells can proliferate without accumulating the genetic damage that drives the initiation and progression of cancer. A classic example of genetic instability in cancer cells is the amplification of segments of the genome in a normally diploid cell type that increases the copy number and thus the expression of oncogenes. However, those who study cell-cycle control in a developmental context know that these types of events are often a normal part of development. This paper by the Orr-Weaver laboratory furthered our understanding of developmentally regulated gene amplification that occurs in Drosophila ovaries. The authors used competitive genome hybridization to a cDNA microarray to identify new follicle cell amplicons required for egg production. This work set the stage for using modern genomic techniques to comprehensively identify gene amplification events that drive the synthesis of large amounts of gene product needed to produce certain structures during development. This PaperPick refers to Gene amplification as a developmental strategy: isolation of two developmental amplicons in Drosophila , by Julie M. Claycomb, Matt Benasutti, Giovanni Bosco, Douglas D. Fenger, and Terry L. Orr-Weaver, published in January 2004. Video Abstract Dr. Orr-Weaver discusses her group's work on regulatory programs that amplify (or suppress) overreplication of cell-type-appropriate genes in the Drosophila embryo.

Sphingosine kinase 1 overexpression is regulated by signaling through PI3K, AKT2, and mTOR in imatinib-resistant chronic myeloid leukemia cells

As a better understanding of the molecular basis of carcinogenesis has emerged, oncogene-specific cell-signaling pathways have been successfully targeted to treat human malignances. Despite impressive advances in oncogene-directed therapeutics, genetic instability in cancer cells often manifest acquired resistance. This is particularly noted in the use of tyrosine kinase inhibitors therapies and not more evident than for chronic myeloid leukemia. Therefore, it is of great importance to understand the molecular mechanisms affecting cancer cell sensitivity and resistance to tyrosine kinase inhibitors. In this study, we used continuous exposure to stepwise increasing concentrations of imatinib (0.6-1 μM) to select imatinib-resistant K562 cells. Expression of BCR-ABL increased both at RNA and protein levels in imatinib-resistant cell lines. Furthermore, expression levels of sphingosine kinase 1 (SphK1) were increased significantly in resistant cells, channeling sphingoid bases to the SphK1 pathway and activating sphingosine-1-phosphate-dependent tyrosine phosphorylation pathways that include the adaptor protein Crk. The partial inhibition of SphK1 activity by N,N-dimethylsphingosine or expression by small interfering RNA increased sensitivity to imatinib-induced apoptosis in resistant cells and returned BCR-ABL to baseline levels. To determine the resistance mechanism-induced SphK1 upregulation, we used pharmacological inhibitors of the phosphoinositide 3-kinase/AKT/mammalian target of rapamycin signaling pathway and observed robust downmodulation of SphK1 expression and activity when AKT2, but not AKT1 or AKT3, was suppressed. These results demonstrate that SphK1 is upregulated in imatinib-resistant K562 cells by a pathway contingent on a phosphoinositide 3-kinase/AKT2/mammalian target of rapamycin signaling pathway. We propose that SphK1 plays an important role in development of acquired resistance to imatinib in chronic myeloid leukemia cell lines.

Cancer Genomes Evolve by Pulverizing Single Chromosomes

A report in this issue describes "chromothripsis," a new mechanism for genetic instability in cancer cells. Chromothripsis appears to be a cataclysmic event in which a single chromosome is fragmented and then reassembled. The phenomenon raises important questions of how chromosome rearrangements can be confined to defined genome segments.

Editorial [Targeting Genetic Instability in Cancer Cells (Guest Editor: Francesco Colotta)

Editorial [Targeting Genetic Instability in Cancer Cells (Guest Editor: Francesco Colotta)

Alterations in p16 and p53 genes and chromosomal findings in patients with lung cancer: Fluorescence in situ hybridization and cytogenetic studies

Background: Chromosomal aberrations and instability of gene(s) are two factors related to the genetic instability of cancer cells. A loss of the tumor-suppressor function of the genes p16 and p53 is the most common event leading to the development of human cancers. Carcinoma of the lung is the leading cause of cancer deaths in the world. Chromosomal abnormalities in lung cancer may provide a valuable clue to the identification of target loci and culminate in a successful search for the major genes. The aim of this study was to investigate (i) alterations of the p16 and p53 genes and (ii) chromosomal aberrations in patients with small cell and non-small cell lung cancer by fluorescence in situ hybridization (FISH) and cytogenetic studies. Methods: We carried out cytogenetic analysis by Giemsa-banding in 18 cases. FISH probes for the p16 and p53 genes were also used on interphase nuclei to screen the alterations in these genes in lung cancer (LC). Results: We observed a high frequency of losses of the p16 – in 8/18 (44%) – and p53 – in 7/18 (39%) – genes in the cases with LC. A total of 18 patients showed predominantly numerical and structural aberrations. Among these two types, structural aberrations predominated and usually consisted of deletions, breaks, and fragilities in various chromosomes. Both structural and numerical changes were observed in almost all patients. Chromosomes 3 and 1 were found to be most frequently involved in structural abnormalities, followed by chromosomes 6, 9, and 8. Autosomal aneuploidies were also observed to be the most frequent (chromosomes 22, 19, 18, 20, 9, and 17), followed by those of the X and Y chromosomes. The expression of fragile sites was also found to be significantly higher in seven chromosomal regions: 3p14, 1q21, 1q12, 6q26, 9q13, 8q22, and 8q24. Conclusion: Our data confirmed that DNA damage and genomic instability may be factors contributing to the mutation profile and development of lung cancer. The patients who developed lung cancer showed a high frequency of loss of both p16 and p53, in addition to chromosomal aberrations. Tobacco could be a major carcinogenic factor in lung-cancer progression. The loss of p16 and p53, and increased incidence of autosomal aneuploidy and chromatid breaks, along with other chromosomal alterations, can contribute to the progression of the disease.

Enhanced specific delivery and targeting of oncolytic Sindbis viral vectors by modulating vascular leakiness in tumor

SummaryGenetic instability of cancer cells generates resistance after initial responses to chemotherapeutic agents. Several oncolytic viruses have been designed to exploit specific signatures of cancer cells, such as important surface markers or pivotal signaling pathways for selective replication. It is less likely for cancer cells to develop resistance given that mutations in these cancer signatures would negatively impact tumor growth and survival. However, as oncolytic viral vectors are large particles, they suffer from inefficient extravasation from tumor blood vessels. For larger particles, such as viral vectors, their ability to reach cancer cells is an important consideration in achieving specific oncolytic targeting and potential vector replication. Our previous studies indicated that the Sindbis viral vectors target tumor cells via the laminin receptor (LAMR). Here, we present evidence that modulating tumor vascular leakiness, using VEGF and/or metronomic chemotherapy regimens significantly enhances tumor vascular permeability and directly enhances oncolytic Sindbis vector targeting in tumor models. Since host-derived vascular endothelium cells are genetically stable and less likely to develop resistance to chemotherapeutics, a combined metronomic chemotherapeutics and oncolytic viruses regimen should provide a new approach for cancer therapy. This mechanism could explain the synergistic treatment outcomes observed in clinical trials of combined therapies.