P53-defective Tumor Cells Research Articles

Replication-competent influenza virus with a protein-responsive multiplication ability

Applications of influenza A viruses (IAV) for virotherapy and biotechnology have accelerated substantially with the development of reverse genetic technology and advances in the understanding of packaging signals. While the use of a replication-competent IAV is particularly promising, owing to its efficient transmission to organ depths with high infectivity, there is also a risk that its multiplication cannot be controlled in a cell-type-specific manner, causing an infectious disease. Therefore, here a simple and effective replication-competent IAV-based cell-targeting system has been developed. It was demonstrated that the activity of the ribonucleoprotein complex (RNP) of IAV could be regulated by the interaction between the endogenous protein and a nanobody fused to the subunit of RNA-dependent RNA polymerase (RdRp). To validate the feasibility of the method, it was demonstrated that RNP containing RdRp fused with Nb139, a nanobody against p53, is inactive in HEK293T cells expressing endogenous p53, but active in p53-defective Saos-2 cells. Finally, a replication-competent IAV was successfully generated that multiplies only in p53-defective tumor cells and an IAV vector was developed that can deliver a foreign gene in cell type-specific manner. The method is flexible because the nanobody can be easily altered to target a different cell type, offering a valuable platform for virotherapy and biotechnology.

AZD1775 Increases Sensitivity to Olaparib and Gemcitabine in Cancer Cells with p53 Mutations.

Tumor suppressor p53 is responsible for enforcing cell cycle checkpoints at G1/S and G2/M in response to DNA damage, thereby allowing both normal and tumor cells to repair DNA before entering S and M. However, tumor cells with absent or mutated p53 are able to activate alternative signaling pathways that maintain the G2/M checkpoint, which becomes uniquely critical for the survival of such tumor cells. We hypothesized that abrogation of the G2 checkpoint might preferentially sensitize p53-defective tumor cells to DNA-damaging agents and spare normal cells with intact p53 function. The tyrosine kinase WEE1 regulates cdc2 activity at the G2/M checkpoint and prevents entry into mitosis in response to DNA damage or stalled DNA replication. AZD1775 is a WEE1 inhibitor that overrides and opens the G2/M checkpoint by preventing WEE1-mediated phosphorylation of cdc2 at tyrosine 15. In this study, we assessed the effect of AZD1775 on endometrial and ovarian cancer cells in the presence of two DNA damaging agents, the PARP1 inhibitor, olaparib, and the chemotherapeutic agent, gemcitabine. We show that AZD1775 alone is effective as a therapeutic agent against some p53 mutated cell models. Moreover, the combination of AZD1775 with olaparib or gemcitabine is synergistic in cells with mutant p53 and constitutes a new approach that should be considered in the treatment of advanced and recurrent gynecologic cancer.

Abstract B32: Loss of MK2 in tumor stroma promotes aggressive tumor development in an advanced stage lung cancer model

Abstract MAPKAP Kinase-2 (MK2) is a serine/threonine protein kinase that is activated by p38MAPK in response to a variety of stresses including DNA damage, osmotic shock, thermal injury, and inflammation/innate immune signaling downstream of TLR4 activation. MK2 controls the synthesis of many pro-inflammatory cytokines, modulates the actin cytoskeleton in stressed cells, and is responsible for maintaining both the G1/S and G2/M cell cycle checkpoints in p53-defective tumor cells after genotoxic damage. We recently created a “Cre-reversible" MK2 knock-out mouse model that allows the function of MK2 within either the tumor cells or within the tumor microenvironment to be studied independently. We found that MK2-deficient tumor cells in an MK2 wild-type stroma were dramatically sensitized to the anti-tumor effects of cisplatinum and doxorubicin (Morandell et al., Cell Reports, 2013). Here, we report that loss of MK2 in the tumor stroma with preservation of MK2 in tumor cells results in an aggressive tumor-promoting phenotype in an immuno-competent transplant K-RasG12D/p53-/- model of NSCLC. A similar tumor aggressive phenotype was observed in Tie2-Cre-driven endothelial and bone marrow-specific MK2-knockout mice compared to wild-type controls. Tumor-bearing mice with MK2-null stroma display increased total levels of MIP-1α, G-CSF, GM-CSF and IL-6 in the lung. Myeloperoxidase immunohistochemistry reveals enhanced tumor infiltration by neutrophils in these stromal MK2-deficient animals, consistent with the cytokine profile. In contrast with loss of MK2 in the stroma, direct activation of the p38MAPK/MK2 pathway in the tumor microenvironment by weekly intra-tracheal LPS exposure dramatically reduces tumor formation in an autochthonous K-RasG12D/p53-/- murine NSCLC model. Collectively, these results suggest that MK2 signaling in the endothelium and/or immune cells suppresses NSCLC tumor progression and modulates the inflammatory milieu in the tumor microenvironment. Citation Format: Ganapathy Sriram, Lucia Suarez-Lopez, Michael B. Yaffe. Loss of MK2 in tumor stroma promotes aggressive tumor development in an advanced stage lung cancer model. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B32.

MK-8776, a novel chk1 kinase inhibitor, radiosensitizes p53-defective human tumor cells.

Radiotherapy is commonly used to treat a variety of solid tumors but improvements in the therapeutic ratio are sorely needed. The aim of this study was to assess the Chk1 kinase inhibitor, MK-8776, for its ability to radiosensitize human tumor cells. Cells derived from NSCLC and HNSCC cancers were tested for radiosensitization by MK-8776. The ability of MK-8776 to abrogate the radiation-induced G2 block was determined using flow cytometry. Effects on repair of radiation-induced DNA double strand breaks (DSBs) were determined on the basis of rad51, γ-H2AX and 53BP1 foci. Clonogenic survival analyses indicated that MK-8776 radiosensitized p53-defective tumor cells but not lines with wild-type p53. Abrogation of the G2 block was evident in both p53-defective cells and p53 wild-type lines indicating no correlation with radiosensitization. However, only p53-defective cells entered mitosis harboring unrepaired DSBs. MK-8776 appeared to inhibit repair of radiation-induced DSBs at early times after irradiation. A comparison of MK-8776 to the wee1 inhibitor, MK-1775, suggested both similarities and differences in their activities. In conclusion, MK-8776 radiosensitizes tumor cells by mechanisms that include abrogation of the G2 block and inhibition of DSB repair. Our findings support the clinical evaluation of MK-8776 in combination with radiation.

DNA Damage Activates a Spatially Distinct Late Cytoplasmic Cell-Cycle Checkpoint Network Controlled by MK2-Mediated RNA Stabilization

Following genotoxic stress, cells activate a complex kinase-based signaling network to arrest the cell cycle and initiate DNA repair. p53-defective tumor cells rewire their checkpoint response and become dependent on the p38/MK2 pathway for survival after DNA damage, despite a functional ATR-Chk1 pathway. We used functional genetics to dissect the contributions of Chk1 and MK2 to checkpoint control. We show that nuclear Chk1 activity is essential to establish a G(2)/M checkpoint, while cytoplasmic MK2 activity is critical for prolonged checkpoint maintenance through a process of posttranscriptional mRNA stabilization. Following DNA damage, the p38/MK2 complex relocalizes from nucleus to cytoplasm where MK2 phosphorylates hnRNPA0, to stabilize Gadd45α mRNA, while p38 phosphorylates and releases the translational inhibitor TIAR. In addition, MK2 phosphorylates PARN, blocking Gadd45α mRNA degradation. Gadd45α functions within a positive feedback loop, sustaining the MK2-dependent cytoplasmic sequestration of Cdc25B/C to block mitotic entry in the presence of unrepaired DNA damage. Our findings demonstrate a critical role for the MK2 pathway in the posttranscriptional regulation of gene expression as part of the DNA damage response in cancer cells.

MOS, aneuploidy and the ploidy cycle of cancer cells

After DNA or spindle damage, p53-defective tumor cells undergo a complex cycle of reversible polyploidy. How this process occurs and more importantly, why, has recently become the focus of several research groups, prompting this review in which we discuss two related phenomena that accompany the reversible polyploidy of tumor cells: the induction of meiosis genes such as MOS and the decrease in genomic instability observed during the reversion from polyploidy to para-diploidy. The reversible polyploidy likely provides the means through which the balance between increased chromosome instability (CIN), driving genetic variation and decreased CIN, necessary for perpetuating these malignant clones, is maintained. These concepts are integrated with recent findings that many meiotic and self-renewal genes become activated during reversible polyploidy and lead us to the hypothesis that tumor cell immortality may be achieved through germline-like transmission.

Abstract SY15-03: Systems biology of DNA damage and repair

Abstract Post-translational modifications such as protein phosphorylation and acetylation, together with binding of the modified proteins to modular signaling domains (i.e. 14-3-3 proteins, WW domains, FHA domains, Polo-box domains, and BRCT domains) function together within signaling networks to control the cellular response to DNA damage. How signals emerging from these pathways are integrated and processed as a network is unclear. To address this, we have been developing systems models of signaling where kinase activities, protein phosphorylation, binding of substrates to phosphoserine/threonine binding domains, and cellular responses including cell cycle arrest and apoptosis are quantitatively measured at densely sampled points in time, and correlated mathematically using partial least squares regression/principal components analysis, or step-wise regression methods. We have used this to approach to map context-dependent signaling events that control the fate of individual cells after genotoxic stress. In addition to the ATM/Chk2 and ATR/Chk1 pathways that contribute to these responses, we have used this approach to identify a third DNA damage signaling pathway mediated by p38 MAPK-activation of MAPKAP Kinase-2 that is required for p53-deficient, but not for p53-proficient, tumor cell survival after DNA damage. In response to cisplatin exposure, MAPKAP Kinase-2 is required for Cdc25A destabilization and activation of the G1 and intra-S checkpoints, while in response to doxorubicin or camptothecin treatment, MAPKAP Kinase-2 is required for targeting of Cdc25B to 14-3-3, and activation of the G2/M checkpoint. MAPKAP Kinase-2 depletion abolishes these cell cycle checkpoints in p53-defective tumor cells, sensitizes them to chemotherapy in culture, and induces dramatic tumor regression after low-dose chemotherapy in a murine xenograft tumor model. No such MAPKAP Kinase-2 dependence is seen in p53-proficient tumors. Chk1 responds to the same genotoxic stresses that activate MAPKAP Kinase-2, and Chk1 is activated normally in the MAPKAP Kinase-2-depleted cells that display aberrant checkpoint function. Thus, MAPKAP Kinase-2 and Chk1 function together as a molecular ‘AND’ gate that is required to integrate DNA damage signals to control cell cycle progression and prevent mitotic catastrophe within tumors. The biological basis for the ‘AND’ gate function of Chk1 and MAPKAP Kinase-2 is directly related to the subcellular context in which each kinase functions. ATM is second molecular target whose inhibition has been postulated to sensitize tumors to chemotherapy. Using in vitro cell culture models together with murine xenografts and Eμ-myc driven lymphomas, we show that ATM inhibition can result in either pronounced tumor resistance or sensitization to DNA damaging chemotherapy, determined solely by the underlying state of the p53 pathway. We show that ATM-deficient p53-wild type tumors that are resistant to chemotherapy can be re-sensitized by inhibition of DNA-PK. These findings for MAPKAP Kinase-2 , ATM and DNA-PK demonstrate how signals from the DNA damage network are re-routed in p53-defective tumor cells, and show how pathway- and network-focused diagnostics can be used to successfully predict therapeutic outcome in human cancer treatment. Citation Format: Michael B. Yaffe. Systems biology of DNA damage and repair [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY15-03

Abstract 690: Estrogen receptor beta (ERβ) enhances the chemosensitivity of breast cancer cells by altering DNA damage response signaling

Abstract Estrogen Receptor beta (ERβ) is a mediator of estrogen signaling that regulates growth in mammary gland and is involved in breast cancer. In response to DNA damage most tumor cells due to disruptions in the p53 pathway and consequent loss of G1 cell cycle arrest are entirely dependent in S and G2/M checkpoints to maintain genomic integrity. Inhibition of these checkpoints following DNA damage induces selective killing of p53-defective tumor cells. We show that expression of ERβ caused abrogation of Cdc25C-mediated G2/M checkpoint following cisplatin and doxorubicin exposure selectively in p53-deficient cells, resulting in mitotic catastrophe and decreased cancer cell survival. The loss of G2/M checkpoint observed was due to ERβ-mediated inhibition of the DNA damage-induced expression of brca1. ERβ by modulating DNA damage response signaling seems to selectively enhance the chemosensitivity of tumor cells. This suggests that co-operation of compounds that specifically induce the ERβ signaling with DNA damaging chemotherapeutic drugs may represent an attractive new cancer therapeutic strategy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 690.

P53-based cancer therapies: is defective p53 the Achilles heel of the tumor?

The tumor suppressor protein p53 plays a pivotal role in the DNA damage response and is defective in >50% of human tumors, which has generated substantial interest in developing p53-targeted cancer therapies. Various therapeutic rationales targeting p53 are currently under investigation including attempts to both activate and inhibit p53. Elevation of p53 can be achieved by either reintroducing an exogenous p53 gene or by blocking its association with its negative regulator hDM2. An alternate approach involves reverting mutant p53 to its wild-type conformation. Inhibition of p53 activity can be achieved either by preventing p53-mediated gene expression or by inhibiting the mitochondrial pro-apoptotic interactions of p53. These approaches are based on the concept that activation of p53 in a tumor is cytotoxic while inhibition of p53 in normal cells will protect the patient. However, activation of p53 also induces cell cycle arrest that can protect most normal cells from DNA damage, and this is the reason why many p53-defective tumors are more sensitive to DNA damage. The development of cell cycle checkpoint inhibitors to abrogate DNA damage-induced arrest builds on this observation as p53-defective cells appear particularly sensitive. Thus, normal cells are protected from premature entry into mitosis and the subsequent mitotic catastrophe induced by checkpoint inhibitors, while p53-defective tumor cells are destroyed. These contradictory approaches must be resolved if we are to take full advantage of the frequent p53 defect in tumors.

P53-Defective Tumors With a Functional Apoptosome-Mediated Pathway: A New Therapeutic Target

Although cancer cells appear to maintain the machinery for intrinsic apoptosis, defects in the pathway develop during malignant transformation, preventing apoptosis from occurring. How to specifically induce apoptosis in cancer cells remains unclear. We determined the apoptosome activity and p53 status of normal human cells and of lung, colon, stomach, brain, and breast cancer cells by measuring cytochrome c-dependent caspase activation and by DNA sequencing, respectively, and we used COMPARE analysis to identify apoptosome-specific agonists. We compared cell death, cytochrome c release, and caspase activation in NCI-H23 (lung cancer), HCT-15 (colon cancer), and SF268 (brain cancer) cells treated with Triacsin c, an inhibitor of acyl-CoA synthetase (ACS), or with vehicle. The cells were mock, transiently, or stably transfected with genes for Triacsin c-resistant ACSL5, dominant negative caspase-9, or apoptotic protease activating factor-1 knockdown. We measured ACS activity and levels of cardiolipin, a mitochondrial phospholipid, in mock and ACSL5-transduced SF268 cells. Nude mice carrying NCI-H23 xenograft tumors (n = 10) were treated with Triacsin c or vehicle, and xenograft tumor growth was assessed. Groups were compared using two-sided Student t tests. Of 21 p53-defective tumor cell lines analyzed, 17 had higher apoptosome activity than did normal cells. Triacsin c selectively induced apoptosome-mediated death in tumor cells (caspase activity of Triacsin c-treated versus untreated SF268 cells; means = 1020% and 100%, respectively; difference = 920%, 95% CI = 900% to 940%; P<.001). Expression of ACSL5 suppressed Triacsin c-induced cytochrome c release and subsequent cell death (cell survival of Triacsin c-treated mock- versus ACSL5-transduced SF268 cells; means = 40% and 83%, respectively; difference = 43%, 95% CI = 39% to 47%; P<.001). ACS was also essential to the maintenance of cardiolipin levels. Finally, Triacsin c suppressed growth of xenograft tumors (relative tumor volume on day 21 of Triacsin c-treated versus untreated mice; means = 4.6 and 9.6, respectively; difference = 5.0, 95% CI = 2.1 to 7.9; P = .006). Many p53-defective tumors retain activity of the apoptosome, which is therefore a potential target for cancer chemotherapy. Inhibition of ACS may be a novel strategy to induce the death of p53-defective tumor cells.

E1A Activates Transcription of p73 and Noxa to Induce Apoptosis

p73, a member of the p53 family of proteins, transcriptionally activates a number of genes involved in the control of cell cycle and apoptosis. Overexpression of p73 was detected in a large number of primary head and neck cancers, and in the established cell lines examined, these all contained inactivating p53 mutations. The significance of p73 overexpression in the pathogenesis of head and neck cancer is currently unclear. We have shown that the expression of adenovirus 5 E1A in a panel of head and neck cancer cell lines induces apoptosis independently of their p53 status. In this study we examined the role of p73 and its transcriptional targets in E1A-mediated induction of apoptosis. E1A expression resulted in significant activation of the TAp73 promoter but had no effect on the alternative, DeltaNp73 promoter. E1A also increased expression of endogenous TAp73 mRNA and protein. E1A mutants lacking the p300- and/or pRB-binding sites showed reduced ability to activate the TAp73 promoter. Additionally, mutations in the E2F1-binding sites in the TAp73 promoter impaired activation by E1A. Importantly, expression of the 13S isoform of E1A substantially induced the p53 apoptotic target Noxa in several p53-deficient cancer cell lines. Our results indicate that E1A activation of p73 and the p53 apoptotic target Noxa can occur in the absence of a functional p53. This activation is likely to play a key role in the mechanism of p53-independent apoptosis induced by E1A in some cancers and may provide an avenue for future cancer therapies.

Late viral RNA export, rather than p53 inactivation, determines ONYX-015 tumor selectivity

ONYX-015 is an adenovirus that lacks the E1B-55K gene product for p53 degradation. Thus, ONYX-015 was conceived as an oncolytic virus that would selectively replicate in p53-defective tumor cells. Here we show that loss of E1B-55K leads to the induction, but not the activation, of p53 in ONYX-015-infected primary cells. We use a novel adenovirus mutant, ONYX-053, to demonstrate that loss of E1B-55K-mediated late viral RNA export, rather than p53 degradation, restricts ONYX-015 replication in primary cells. In contrast, we show that tumor cells that support ONYX-015 replication provide the RNA export function of E1B-55K. These data reveal that tumor cells have altered mechanisms for RNA export and resolve the controversial role of p53 in governing ONYX-015 oncolytic selectivity.

Cell cycle checkpoints and their impact on anticancer therapeutic strategies.

Cells contain numerous pathways designed to protect them from the genomic instability or toxicity that can result when their DNA is damaged. The p53 tumor suppressor is particularly important for regulating passage through G1 phase of the cell cycle, while other checkpoint regulators are important for arrest in S and G2 phase. Tumor cells often exhibit defects in these checkpoint proteins, which can lead to hypersensitivity; proteins in this class include ataxia-telangiectasia mutatated (ATM), Meiotic recanbination 11 (Mre11), Nijmegen breakage syndrome 1 (Nbs 1), breast cancer susceptibility genes 1 and 2 (BRCA1), and (BRCA2). Consequently, tumors should be assessed for these specific defects, and specific therapy prescribed that has high probability of inducing response. Tumors defective in p53 are frequently considered resistant to apoptosis, yet this defect also provides an opportunity for targeted therapy. When their DNA is damaged, p53-defective tumor cells preferentially arrest in S or G2 phase where they are susceptible to checkpoint inhibitors such as caffeine and UCN-01. These inhibitors preferentially abrogate cell cycle arrest in p53-defective cells, driving them through a lethal mitosis. Wild type p53 can prevent abrogation of arrest by elevating levels of p21(waf1) and decreasing levels of cyclins A and B. During tumorigenesis, tumor cells frequently loose checkpoint controls and this facilitates the development of the tumor. However, these defects also represent an Achilles heel that can be targeted to improve current therapeutic strategies.

Chemosensitivity linked to p73 function

Most chemotherapeutic agents induce DNA damage, leading to p53 accumulation and apoptosis. The factors that determine chemosensitivity in p53-defective tumor cells are poorly understood. We found that the p53 family member p73 is induced by a wide variety of chemotherapeutic drugs. Blocking p73 function with a dominant-negative mutant, siRNA, or homologous recombination led to chemoresistance of human tumor cells and engineered transformed cells, irrespective of p53 status. Mutant p53 can inactivate p73 and downregulation of mutant p53 enhanced chemosensitivity. These findings indicate that p73 is a determinant of chemotherapeutic efficacy in humans.

1267. Targeting Gene Expression to the p53 Defective Tumor Cells

1267. Targeting Gene Expression to the p53 Defective Tumor Cells

Viral oncoproteins discriminate between p53 and the p53 homolog p73.

p73 is a recently identified member of the p53 family. Previously it was shown that p73 can, when overproduced in p53-defective tumor cells, activate p53-responsive promoters and induce apoptosis. In this report we describe the generation of anti-p73 monoclonal antibodies and confirm that two previously described p73 isoforms are produced in mammalian cells. Furthermore, we show that these two isoforms can bind to canonical p53 DNA-binding sites in electrophoretic mobility shift assays. Despite the high degree of similarity between p53 and p73, we found that adenovirus E1B 55K, simian virus 40 T, and human papillomavirus E6 do not physically interact with p73. The observation that viral oncoproteins discriminate between p53 and p73 suggests that the functions of these two proteins may differ under physiological conditions. Furthermore, they suggest that inactivation of p73 may not be required for transformation.

Lisofylline Sensitizes p53 Mutant Human Ovarian Carcinoma Cells to the Cytotoxic Effects ofcis-Diamminedichloroplatinum(II)

A novel approach to counteracting drug resistance is the development of nontoxic agents that are able to preferentially increase the sensitivity of tumor cells to the cytotoxicity of chemotherapy. The possibility that such an agent could be directed specifically against p53-defective tumor cells led us to study the new methylxanthine, Lisofylline, for its ability to sensitize ovary cancer cells tocis-diamminedichloroplatinum(II) (CDDP). In cell lines lacking functional p53 (SKOV3, SKOV3 CDDP-resistant, OVCAR3, and OVCAR432) Lisofylline (20–100 μM) enhanced the cytotoxicity of CDDP by approximately 50% as measured by the Alamar blue vital dye indicator assay. LSF had no effect on p53 wild-type cell lines: OVCAR 420,429, and 433. Restoration of wild-type p53 phenotype by transfection of SKOV3 cells with a p53 cDNA expression vector showed reversal of sensitization by Lisofylline to CDDP cytotoxicity. While sensitization to DNA damaging agents by other methylxanthines is related to an abrogation of G2 delay, FACS data found no loss of CDDP-induced G2 block in the cell lines, demonstrating that Lisofylline enhanced sensitization. Cell death was examined by quantitative fluorescence microscopy but no increase in apoptosis attributable to Lisofylline exposure was observed. Our results show that the combination of CDDPand Lisofylline preferentially sensitizes p53-defective cancer cells to the cytotoxic effect of CDDP by a yet undetermined mechanism.

The role of inhibitory phosphorylation of CDC2 following DNA replication block and radiation-induced damage in human cells.

It has been suggested that the survival response of p53 defective tumor cells to agents that inhibit DNA replication or damage DNA may be largely dependent on cell cycle checkpoints that regulate the onset of mitosis. In human cells, the mitosis-inducing kinase CDC2/cyclin B is inhibited by phosphorylation of threonine-14 and tyrosine-15, but the roles of these phosphorylations in enforcing checkpoints is not known. We have investigated the situation in a human cervical carcinoma cell line (HeLa cells) and found that low level expression of a mutant nonphosphorylatable form of CDC2 abrogates regulation of the endogenous CDC2/cyclin B. Disruption of this pathway is toxic and renders cells highly sensitive to killing by DNA damage or by inhibition of DNA replication. These findings establish the importance of inhibitory phosphorylation of CDC2 in the survival mechanism used by human cells when exposed to some of the most common forms of anticancer therapy.