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- TRIB2 confers resistance to anti-cancer therapy by activating the serine/threonine protein kinase AKTPublication . Hill, Richard; Madureira, Patricia; Ferreira, Bibiana; Baptista, Inês; Machado, S.; Colaco, Laura; dos Santos, Marta; Liu, Ningshu; Dopazo, Ana; Ugurel, Selma; Adrienn, Angyal; Kiss-Toth, Endre; Isbilen, Murat; Gure, Ali O.; Link, WolfgangIntrinsic and acquired resistance to chemotherapy is the fundamental reason for treatment failure for many cancer patients. The identification of molecular mechanisms involved in drug resistance or sensitization is imperative. Here we report that tribbles homologue 2 (TRIB2) ablates forkhead box O activation and disrupts the p53/MDM2 regulatory axis, conferring resistance to various chemotherapeutics. TRIB2 suppression is exerted via direct interaction with AKT a key signalling protein in cell proliferation, survival and metabolism pathways. Ectopic or intrinsic high expression of TRIB2 induces drug resistance by promoting phospho-AKT (at Ser473) via its COP1 domain. TRIB2 expression is significantly increased in tumour tissues from patients correlating with an increased phosphorylation of AKT, FOXO3a, MDM2 and an impaired therapeutic response. This culminates in an extremely poor clinical outcome. Our study reveals a novel regulatory mechanism underlying drug resistance and suggests that TRIB2 functions as a regulatory component of the PI3K network, activating AKT in cancer cells.
- Genotoxic agents promote the nuclear accumulation of annexin A2: role of annexin A2 in mitigating DNA damagePublication . Madureira, Patricia A.; Hill, Richard; Lee, Patrick W. K.; Waisman, David M.Annexin A2 is an abundant cellular protein that is mainly localized in the cytoplasm and plasma membrane, however a small population has been found in the nucleus, suggesting a nuclear function for the protein. Annexin A2 possesses a nuclear export sequence (NES) and inhibition of the NES is sufficient to cause nuclear accumulation. Here we show that annexin A2 accumulates in the nucleus in response to genotoxic agents including gamma-radiation, UV radiation, etoposide and chromium VI and that this event is mediated by the nuclear export sequence of annexin A2. Nuclear accumulation of annexin A2 is blocked by the antioxidant agent N-acetyl cysteine (NAC) and stimulated by hydrogen peroxide (H2O2), suggesting that this is a reactive oxygen species dependent event. In response to genotoxic agents, cells depleted of annexin A2 show enhanced phospho-histone H2AX and p53 levels, increased numbers of p53-binding protein 1 nuclear foci and increased levels of nuclear 8-oxo-2'-deoxyguanine, suggesting that annexin A2 plays a role in protecting DNA from damage. This is the first report showing the nuclear translocation of annexin A2 in response to genotoxic agents and its role in mitigating DNA damage.
- The role of hypoxia in glioblastoma invasionPublication . Monteiro, Ana; Hill, Richard; Pilkington, Geoffrey; A Madureira, PatriciaGlioblastoma multiforme (GBM), a grade IV astrocytoma, is the most common and deadly type of primary malignant brain tumor, with a patient's median survival rate ranging from 15 to 17 months. The current treatment for GBM involves tumor resection surgery based on MRI image analysis, followed by radiotherapy and treatment with temozolomide. However, the gradual development of tumor resistance to temozolomide is frequent in GBM patients leading to subsequent tumor regrowth/relapse. For this reason, the development of more effective therapeutic approaches for GBM is of critical importance. Low tumor oxygenation, also known as hypoxia, constitutes a major concern for GBM patients, since it promotes cancer cell spreading (invasion) into the healthy brain tissue in order to evade this adverse microenvironment. Tumor invasion not only constitutes a major obstacle to surgery, radiotherapy, and chemotherapy, but it is also the main cause of death in GBM patients. Understanding how hypoxia triggers the GBM cells to become invasive is paramount to developing novel and more effective therapies against this devastating disease. In this review, we will present a comprehensive examination of the available literature focused on investigating how GBM hypoxia triggers an invasive cancer cell phenotype and the role of these invasive proteins in GBM progression.
- Components and regulation of nuclear transport processesPublication . Cautain, Bastien; Hill, Richard; de Pedro, Nuria; Link, WolfgangThe spatial separation of DNA replication and gene transcription in the nucleus and protein translation in the cytoplasm is a uniform principle of eukaryotic cells. This compartmentalization imposes a requirement for a transport network of macromolecules to shuttle these components in and out of the nucleus. This nucleo-cytoplasmic transport of macromolecules is critical for both cell physiology and pathology. Consequently, investigating its regulation and disease-associated alterations can reveal novel therapeutic approaches to fight human diseases, such as cancer or viral infection. The characterization of the nuclear pore complex, the identification of transport signals and transport receptors, as well as the characterization of the Ran system (providing the energy source for efficient cargo transport) has greatly facilitated our understanding of the components, mechanisms and regulation of the nucleo-cytoplasmic transport of proteins in our cells. Here we review this knowledge with a specific emphasis on the selection of disease-relevant molecular targets for potential therapeutic intervention.
- Retraction note: TRIB2 confers resistance to anti-cancer therapy by activating the serine/threonine protein kinase AKTPublication . Hill, Richard; Madureira, Patricia; Ferreira, Bibiana; Baptista, Ines; Machado, Susana; Colaҫo, Laura; dos Santos, Marta; Liu, Ningshu; Dopazo, Ana; Ugurel, Selma; Adrienn, Angyal; Kiss-Toth, Endre; Isbilen, Murat; Gure, Ali O.; Link, WolfgangThe authors have retracted this article as it has come to their attention that several images were inappropriately processed and duplicated in multiple figures. In particular, the data were duplicated, and in some cases inverted, across several panels in Figures 2c, 2b, 3d and Supplementary Figure 5. Erroneous data were also included in Figure 2e, Supplementary Figure 1 and Supplementary Figure 8. We apologize to the scientific community for any confusion this article may have caused. Richard Hill, Patricia Madureira, Bibiana I. Ferreira, Susana Machado, Ana Dopazo, Selma Ugurel, Endre Kiss-Toth, Murat isbilen and Wolfgang Link agree with this retraction. Inês Baptista, Laura Colaço, Marta dos Santos, Ningshu Liu, Angyal Adrienn and Ali O. Gure have not responded to correspondence from the Publisher about this retraction.
- Methylseleninic acid promotes antitumour effects via nuclear FOXO3a translocation through Akt inhibitionPublication . Tarrado-Castellarnau, Miriam; Cortes, Roldan; Zanuy, Miriam; Tarrago-Celada, Josep; Polat, Ibrahim H.; Hill, Richard; Fan, Teresa W. M.; Link, Wolfgang; Cascante, MartaSelenium supplement has been shown in clinical trials to reduce the risk of different cancers including lung carcinoma. Previous studies reported that the antiproliferative and pro-apoptotic activities of methylseleninic acid (MSA) in cancer cells could be mediated by inhibition of the PI3K pathway. A better understanding of the downstream cellular targets of MSA will provide information on its mechanism of action and will help to optimize its use in combination therapies with PI3K inhibitors. For this study, the effects of MSA on viability, cell cycle, metabolism, apoptosis, protein and mRNA expression, and reactive oxygen species production were analysed in A549 cells. FOXO3a subcellular localization was examined in A549 cells and in stably transfected human osteosarcoma U2foxRELOC cells. Our results demonstrate that MSA induces FOXO3a nuclear translocation in A549 cells and in U2OS cells that stably express GFP-FOXO3a. Interestingly, sodium selenite, another selenium compound, did not induce any significant effects on FOXO3a translocation despite inducing apoptosis. Single strand break of DNA, disruption of tumour cell metabolic adaptations, decrease in ROS production, and cell cycle arrest in G1 accompanied by induction of apoptosis are late events occurring after 24h of MSA treatment in A549 cells. Our findings suggest that FOXO3a is a relevant mediator of the antiproliferative effects of MSA. This new evidence on the mechanistic action of MSA can open new avenues in exploiting its antitumour properties and in the optimal design of novel combination therapies. We present MSA as a promising chemotherapeutic agent with synergistic antiproliferative effects with cisplatin. (C) 2015 Elsevier Ltd. All rights reserved.
- Gemcitabine-mediated tumour regression and p53-dependent gene expression: implications for colon and pancreatic cancer therapyPublication . Hill, Richard; Rabb, M.; A Madureira, Patricia; Clements, D.; Gujar, S. A.; Waisman, D. M.; Giacomantonio, C. A.; Lee, P. W. K.Gemcitabine is a chemotherapeutic that is widely used for the treatment of a variety of haematological malignancies and has become the standard chemotherapy for the treatment of advanced pancreatic cancer. Combinational gemcitabine regimes (e.g. with doxorubicin) are being tested in clinical trials to treat a variety of cancers, including colon cancer. The limited success of these trials has prompted us to pursue a better understanding of gemcitabine's mechanism of cell killing, which could dramatically improve the therapeutic potential of this agent. For comparison, we included gamma irradiation that triggers robust cell cycle arrest and Cr(VI), which is a highly toxic chemical that induces a robust p53-dependent apoptotic response. Gemcitabine induced a potent p53-dependent apoptosis that correlated with the accumulation of pro-apoptotic proteins such as PUMA and Bax. This is accompanied by a drastic reduction in p2l and 14-3-3 sigma protein levels, thereby significantly sensitizing the cells to apoptosis. In vitro and in vivo studies demonstrated that gemcitabine required PUMA transcription to instigate an apoptotic programme. This was in contrast to Cr(VI)-induced apoptosis that required Bax and was independent of transcription. An examination of clinical colon and pancreatic cancer tissues shows higher p53, p21, 14-3-3 sigma and Bax expression compared with matched normal tissues, yet there is a near absence of PUMA protein. This may explain why gemcitabine shows only limited efficacy in the treatment of these cancers. Our results raise the possibility that targeting the Bax-dependent cell death pathway, rather than the PUMA pathway, could result in significantly improved patient outcome and prognosis for these cancers.
- Targeting nucleocytoplasmic transport in cancer therapyPublication . Hill, Richard; Cautain, Bastien; de Pedro, Nuria; Link, WolfgangThe intracellular location and regulation of proteins within each cell is critically important and is typically deregulated in disease especially cancer. The clinical hypothesis for inhibiting the nucleo-cytoplasmic transport is based on the dependence of certain key proteins within malignant cells. This includes a host of well-characterized tumor suppressor and oncoproteins that require specific localization for their function. This aberrant localization of tumour suppressors and oncoproteins results in their their respective inactivation or over-activation. This incorrect localization occurs actively via the nuclear pore complex that spans the nuclear envelope and is mediated by transport receptors. Accordingly, given the significant need for novel, specific disease treatments, the nuclear envelope and the nuclear transport machinery have emerged as a rational therapeutic target in oncology to restore physiological nucleus/cytoplasmic homeostasis. Recent evidence suggests that this approach might be of substantial therapeutic use. This review summarizes the mechanisms of nucleocytoplasmic transport, its role in cancer biology and the therapeutic potential of targeting this critical cellular process
- DIVERSet JAG compounds inhibit topoisomerase II and are effective against adult and pediatric high-grade gliomasPublication . Howarth, Alison; Simms, Claire; Kerai, Nitesh; Allen, Olivia; Mihajluk, Karina; Madureira, Patricia; Sokratous, Giannis; Cragg, Simon; Lee, Sang Y.; Morley, Andy D.; Ashkan, Keyoumars; Cox, Paul A.; Pilkington, Geoffrey J.; Hill, RichardHigh-grade gliomas (HGGs) are aggressive primary brain tumors with local invasive growth and poor clinical prognosis in both adult and pediatric patients. Clinical response is compounded by resistance to standard frontline antineoplastic agents, an absence of novel therapeutics, and poor in vitro models to evaluate these. We screened a range of recently identified anticancer compounds in conventional adult, pediatric, and new biopsy-derived HGG models. These in vitro lines showed a range of sensitivity to standard chemotherapeutics, with varying expression levels of the prognostic markers hypoxia-induced factor (HIF) 1α and p53. Our evaluation of lead DIVERSet library compounds identified that JAG-6A, a compound that was significantly more potent than temozolomide or etoposide, was effective against HGG models in two-dimensional and three-dimensional systems; mediated this response by the potent inhibition of topoisomerase Iiα; remained effective under normoxic and hypoxic conditions; and displayed limited toxicity to non-neoplastic astrocytes. These data suggest that JAG-6A could be an alternative topoisomerase IIα inhibitor and used for the treatment of HGG.
- A novel phosphatidylinositol 3-kinase (PI3K) inhibitor directs a potent FOXO-dependent, p53-independent cell cycle arrest phenotype characterized by the differential induction of a subset of FOXO-regulated genesPublication . Hill, Richard; Kalathur, Ravi Kiran Reddy; Callejas, Sergio; Colaço, Laura; Brandão, Ricardo; Serelde, Beatriz; Cebriá, Antonio; Blanco-Aparicio, Carmen; Pastor, Joaquín; Futschik, Matthias; Dopazo, Ana; Link, WolfgangIntroduction: The activation of the phosphoinositide 3-kinase (PI3K)/AKT signalling pathway is one the most frequent genetic events in breast cancer, consequently the development of PI3K inhibitors has attracted much attention. Here we evaluate the effect of PI3K inhibition on global gene expression in breast cancer cells. Methods: We used a range of methodologies that include in silico compound analysis, in vitro kinase assays, cell invasion assays, proliferation assays, genome-wide transcription studies (Agilent Technologies full genome arrays), gene set enrichment analysis, quantitative real-time PCR, immunoblotting in addition to chromatin immunoprecipitation. Results: We defined the physico-chemical and the biological properties of ETP-45658, a novel potent PI3K inhibitor. We demonstrated that ETP-45658 potently inhibited cell proliferation within a broad range of human cancer cells, most potently suppressing the growth of breast cancer cells via inhibiting cell cycle. We show that this response is Forkhead box O (FOXO) protein dependent and p53 independent. Our genome-wide microarray analysis revealed that the cell cycle was the most affected biological process after exposure to ETP-45658 (or our control PI3K inhibitor PI-103), that despite the multiple transcription factors that are regulated by the PI3K/AKT signalling cascade, only the binding sites for FOXO transcription factors were significantly enriched and only a subset of all FOXO-dependent genes were induced. This disparity in gene transcription was not due to differential FOXO promoter recruitment. Conclusions: The constitutive activation of PI3Ks and thus the exclusion of FOXO transcription factors from the nucleus is a key feature of breast cancer. Our results presented here highlight that PI3K inhibition activates specific FOXO-dependent genes that mediate cell cycle arrest in breast cancer cells.