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PDF file - 2823K, Supplementary Figure 1. Estrogen metabolites cause DNA double strand breaks in S- phase cells. Supplementary Figure 2. BRCA1 supresses estrogen metabolite mediated DSBs and is required for their repair. Supplementary Figure 3. Estrogen and its metabolites induce DNA DSBs in a dose and time dependent manner. Supplementary Figure 4. BRCA1 suppresses estrogen metabolites induced DNA damage in BRCA1 mutant breast cancer cells. Supplementary Figure 5. BRCA1 and BRCA2 are required for repair of estrogen metabolite induced DSBs in MCF10A and MCF7 breast cells. Supplementary Figure 6. BRCA1, but not BRCA2, suppresses estrogen metabolite mediated DNA damage by repressing CYP1A1 expression in breast cells.

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Germline mutations in BRCA1 predispose carriers to a high incidence of breast and ovarian cancers. BRCA1 functions to maintain genomic stability through critical roles in DNA repair, cell-cycle arrest, and transcriptional control. A major question has been why BRCA1 loss or mutation leads to tumors mainly in estrogen-regulated tissues, given that BRCA1 has essential functions in all cell types. Here, we report that estrogen and estrogen metabolites can cause DNA double-strand breaks (DSB) in estrogen receptor-α–negative breast cells and that BRCA1 is required to repair these DSBs to prevent metabolite-induced genomic instability. We found that BRCA1 also regulates estrogen metabolism and metabolite-mediated DNA damage by repressing the transcription of estrogen-metabolizing enzymes, such as CYP1A1, in breast cells. Finally, we used a knock-in human cell model with a heterozygous BRCA1 pathogenic mutation to show how BRCA1 haploinsufficiency affects these processes. Our findings provide pivotal new insights into why BRCA1 mutation drives the formation of tumors in estrogen-regulated tissues, despite the general role of BRCA1 in DNA repair in all cell types. Cancer Res; 74(10); 2773–84. ©2014 AACR.

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Advancements in the development of targeted therapies have changed the therapeutic paradigm in acute myeloid leukemia (AML) in recent years. The orally available BCL-2 inhibitor venetoclax is effective and well tolerated in combination with either a hypomethylating agent (HMA) or low-dose cytarabine (LDAC). BCL-2 is a anti-apoptotic protein which binds to and sequesters pro-apoptotic proteins such as BAX, BAK, PUMA and NOXA and prevents pro-apoptotic proteins from initiating mitochondrial apoptosis leading to sustained survival in cancer cells. Venetoclax specifically antagonizes BCL-2 protein and allows unbound pro-apoptotic proteins to initiate apoptosis. Venetoclax antagonizes BCL-2 and allows pro-apoptotic proteins to initiate apoptosis. Venetoclax with HMA or LDAC received FDA approval in 2020 for the frontline treatment of AML patients unfit for standard chemotherapy. However, therapeutic resistance is common and there is an urgent need to improve the efficacy and durability of venetoclax in this setting. Combinatory drug approaches allow for targeting of multiple molecular drivers/pathogenic mutations, lower therapeutic concentrations, increased efficacy, and overcoming resistance. Here, we aimed to develop novel synergistic drug combinations with venetoclax to improve venetoclax safety, efficacy and overcome resistance in cell lines with potential clinical applications in AML. Venetoclax IC50 values in eight AML cell lines range from 5 µM highlighting differential response among the panel. THP-1 cells were identified as the most venetoclax resistant cell line with little response observed at any concentration up to 72 hours exposure. Western blot and qPCR analyses demonstrated BCL-2 is ubiquitously expressed across the cell lines. A single agent drug screen of 1,971 FDA approved compounds was performed at two concentrations (100 nM and 1 µM), in THP-1 cells at three timepoints (24, 48, and 72 hours). 364 of 1971 compounds (18.5%) induced >50% cell death and were identified as hits. A secondary screen was performed to investigate the top 180 most effective compounds in combination with venetoclax. Dose response matrices and cell viability data were used to calculate synergy scores. Combinations with synergy scores of >10 were considered synergistic. Many combinations were synergistic at one timepoint (n=117, 65%), others were synergistic at 2 timepoints (n=45, 25%) and few were synergistic at all 3 timepoints (n=5, 2.78%). Validation experiments are currently ongoing to investigate the synergistic relationship of top candidate combinations and identify an optimum dose for further investigations including additional validation experiments, mechanistic studies, and toxicity studies. Citation Format: Ruairidh Harrigan, Evangeline Johnston, Kyle B Matchett, Tony J Bjourson. High-capacity drug screen identification of novel synergistic drug pairs with venetoclax in acute myeloid leukemia [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A02.

Paediatric acute myeloid leukaemia (AML) is a heterogeneous disease characterised by genetics and morphology. The introduction of intensive chemotherapy treatments together with patient stratification and supportive therapy has resulted in a moderate improvement in patient prognosis. However, overall survival rates remain unacceptably poor, with only 65% of patients surviving longer than 5 years. Recently age-specific differences in AML have been identified, highlighting the need for tailored treatments for paediatric patients. Combination therapies have the potential to improve patient prognosis, while minimising harmful side-effects. In the laboratory setting, identifying key combinations from large drug libraries can be resource-intensive, prohibiting discovery and translation into the clinic. To minimise redundancy and maximise discovery, we undertook a multiplex screen of 80 apoptotic-inducing agents in paediatric AML pre-clinical models. The screen was designed using an all-pairs testing algorithm, which ensured that all pairs of compounds could be tested, while minimising the number of wells used. We identified a combination of ABT-737, a Bcl-2 family inhibitor and Purvalanol A, a CDK inhibitor, as a potential targeted therapy for AML patients with an MLL rearrangement and an FLT3-ITD. Our approach has the potential to reduce resource-intensity and time associated with the identification of novel combination therapies.

Tibetans have adapted to the chronic hypoxia of high altitude and display a distinctive suite of physiologic adaptations, including augmented hypoxic ventilatory response and resistance to pulmonary hypertension. Genome-wide studies have consistently identified compelling genetic signatures of natural selection in two genes of the Hypoxia Inducible Factor pathway, PHD2 and HIF2A. The product of the former induces the degradation of the product of the latter. Key issues regarding Tibetan PHD2 are whether it is a gain-of-function or loss-of-function allele, and how it might contribute to high-altitude adaptation. Tibetan PHD2 possesses two amino acid changes, D4E and C127S. We previously showed that in vitro, Tibetan PHD2 is defective in its interaction with p23, a cochaperone of the HSP90 pathway, and we proposed that Tibetan PHD2 is a loss-of-function allele. Here, we report that additional PHD2 mutations at or near Asp-4 or Cys-127 impair interaction with p23 in vitro. We find that mice with the Tibetan Phd2 allele display augmented hypoxic ventilatory response, supporting this loss-of-function proposal. This is phenocopied by mice with a mutation in p23 that abrogates the PHD2:p23 interaction. Hif2a haploinsufficiency, but not the Tibetan Phd2 allele, ameliorates hypoxia-induced increases in right ventricular systolic pressure. The Tibetan Phd2 allele is not associated with hemoglobin levels in mice. We propose that Tibetans possess genetic alterations that both activate and inhibit selective outputs of the HIF pathway to facilitate successful adaptation to the chronic hypoxia of high altitude.

Several histone deacetylase inhibitors including Vorinostat have received FDA approval for the treatment of haematological malignancies. However, data from these trials indicate that Vorinostat has limited efficacy as a monotherapy, prompting the need for rational design of combination therapies. A number of epi-sensitised pathways, including sonic hedgehog (SHH), were identified in AML cells by integration of global patterns of histone H3 lysine 9 (H3K9) acetylation with transcriptomic analysis following Vorinostat-treatment. Direct targeting of the SHH pathway with SANT-1, following Vorinostat induced epi-sensitisation, resulted in synergistic cell death of AML cells. In addition, xenograft studies demonstrated that combination therapy induced a marked reduction in leukemic burden compared to control or single agents. Together, the data supports epi-sensitisation as a potential component of the strategy for the rational development of combination therapies in AML.

Ran is a small ras-related GTPase and is highly expressed in aggressive breast carcinoma. Overexpression induces malignant transformation and drives metastatic growth. We have designed a novel series of anti-Ran-GTPase peptides, which prevents Ran hydrolysis and activation, and although they display effectiveness in silico, peptide activity is suboptimal in vitro due to reduced bioavailability and poor delivery. To overcome this drawback, we delivered an anti-Ran-GTPase peptide using encapsulation in PLGA-based nanoparticles (NP). Formulation variables within a double emulsion solvent evaporation technique were controlled to optimise physicochemical properties. NP were spherical and negatively charged with a mean diameter of 182–277 nm. Peptide integrity and stability were maintained after encapsulation and release kinetics followed a sustained profile. We were interested in the relationship between cellular uptake and poly(ethylene glycol) (PEG) in the NP matrix, with results showing enhanced in vitro uptake with increasing PEG content. Peptide-loaded, pegylated (10% PEG)-PLGA NP induced significant cytotoxic and apoptotic effects in MDA-MB-231 breast cancer cells, with no evidence of similar effects in cells pulsed with free peptide. Western blot analysis showed that encapsulated peptide interfered with the proposed signal transduction pathway of the Ran gene. Our novel blockade peptide prevented Ran activation by blockage of regulator of chromosome condensation 1 (RCC1) following peptide release directly in the cytoplasm once endocytosis of the peptide-loaded nanoparticle has occurred. RCC1 blockage was effective only when a nanoparticulate delivery approach was adopted.

The delivery of anticancer agents to their subcellular sites of action is a significant challenge for effective cancer therapy. Peptides, which are integral to several oncogenic pathways, have significant potential to be utilised as cancer therapeutics due to their selectivity, high potency and lack of normal cell toxicity. Novel Ras protein-Regulator of chromosome condensation 1 (Ran-RCC1) inhibitory peptides designed to interact with Ran, a novel therapeutic target in breast cancer, were delivered by entrapment into polyethylene glycol-poly (lactic-co-glycolic acid) PEG-PLGA polymeric nanoparticles (NPs). A modified double emulsion solvent evaporation technique was used to optimise the physicochemical properties of these peptide-loaded biodegradable NPs. The anti-cancer activity of peptide-loaded NPs was studied in vitro using Ran-expressing metastatic breast (MDA-MB-231) and lung cancer (A549) cell lines, and in vivo using Solid Ehrlich Carcinoma-bearing mice. The anti-metastatic activity of peptide-loaded NPs was investigated using migration, invasion and colony formation assays in vitro. A PEG-PLGA-nanoparticle encapsulating N-terminal peptide showed a pronounced antitumor and anti-metastatic action in lung and breast cancer cells in vitro and caused a significant reduction of tumor volume and associated tumor growth inhibition of breast cancer model in vivo. These findings suggest that the novel inhibitory peptides encapsulated into PEGylated PLGA NPs are delivered effectively to interact and deactivate Ran. This novel Ran-targeting peptide construct shows significant potential for therapy of breast cancer and other cancers mediated by Ran overexpression.

Background Overexpression of the RAN GTP (RAN) gene has been shown to be linked to metastatic activity of MDA-MB231 human breast cancer cells by increasing Ras/MEK/ERK and PI3K/Akt/mTORC1 signalling. The aim of this study was to investigate the potential of polymeric nanoparticles to deliver two novel shRNA sequences, targeted against the RAN gene, to MDA-MB231 cells grown in culture and to assess their effects in a range of biological assays. Methods Biodegradable PLGA nanoparticles, loaded with shRNA-1 and shRNA-4, were fabricated using a double emulsion solvent evaporation technique and characterised for size, zeta potential and polydispersity index before testing on the MDA-MB231 cell line in a range of assays including cell viability, migration, invasion and gene knock down. Results shRNA-loaded nanoparticles were successfully fabricated and delivered to MDA-MB231 cells in culture, where they effectively released their payload, causing a decrease in both cell invasion and cell migration by knocking down RAN gene expression. Conclusion Results indicate the anti-RAN shRNA-loaded nanoparticles deliver and release biological payload to MDA-MB231 cells in culture. This works paves the way for further investigations into the possible use of anti-RAN shRNA-loaded NP formulations for the treatment of breast cancer in vivo.

Insights distilled from integrating multiple big-data or “omic” datasets have revealed functional hierarchies of molecular networks driving tumorigenesis and modifiers of treatment response. Identifying these novel key regulatory and dysregulated elements is now informing personalized medicine. Crucially, although there are many advantages to this approach, there are several key considerations to address. Here, we examine how this big data–led approach is impacting many diverse areas of cancer research, through review of the key presentations given at the Irish Association for Cancer Research Meeting and importantly how the results may be applied to positively affect patient outcomes. Cancer Res; 76(21); 6167–70. ©2016 AACR.

It has been shown previously that cancer cells with an activated oncogenic pathway, including Met activation, require Ran for growth and survival.Here, we show that knockdown of Ran leads to a reduction of Met receptor expression in several breast and lung cancer cell lines. This, in turn suppressed HGF expression and the Met-mediated activation of the Akt pathway, as well as cell adhesion, migration, and invasion. In a cell line model where Met amplification has previously been shown to contribute to gefitinib resistance, Ran knockdown sensitized cells to gefitinib-mediated inhibition of Akt and ERK1/2 phosphorylation and consequently reduced cell proliferation. We further demonstrate that Met reduction-mediated by knockdown of Ran, occurs at the post-transcriptional level, probably via a matrix metalloproteinase. Moreover, the level of immunoreactive Ran and Met are positively associated in human breast cancer specimens, suggesting that a high level of Ran may be a pre-requisite for Met overexpression. Interestingly, a high level of immunoreactive Ran dictates the prognostic significance of Met, indicating that the co-overexpression of Met and Ran may be associated with cancer progression and could be used in combination as a prognostic indicator.

The traditional bench-to-bedside pipeline involves using model systems and patient samples to provide insights into pathways deregulated in cancer. This discovery reveals new biomarkers and therapeutic targets, ultimately stratifying patients and informing cohort-based treatment options. Precision medicine (molecular profiling of individual tumors combined with established clinical-pathological parameters) reveals, in real-time, individual patient's diagnostic and prognostic risk profile, informing tailored and tumor-specific treatment plans. Here we discuss advances in precision medicine presented at the Irish Association for Cancer Research Annual Meeting, highlighting examples where personalized medicine approaches have led to precision discovery in individual tumors, informing customized treatment programs.

There is still much to learn about the support required by postgraduate research students, across academic disciplines, to facilitate successful completion of a research degree. The primary aim of this study was to explore postgraduate medical science research students’ perceptions of academic and mentoring support at different stages during their degree programme. A second aim was to explore the feasibility and acceptability of peer mentoring as a strategy to enhance student support in this population. A mixed method study design was used. Students first completed an online questionnaire, which was then supplemented with focus group discussion to explore emergent findings in greater depth. The main results indicated that the nature and quality of academic supervision support have a significant influence on research student training and development. ‘Functional’ and ‘relationship development’ concepts of supervisory styles were highlighted as important aspects to perceived support in this research population. The main facilitators to enhance academic support were effective communication, project planning and timely feedback. There was a high degree of acceptability for a peer mentoring programme as a complementary approach to enhance student support. Peer mentoring psychosocial functions such as friendship, counselling and career guidance were considered potentially beneficial to enhancing student support. Students also discussed peer coaching as central to their learning and research skill development. This work is a useful starting point to explore perceptions of research student support in the target population. Further work is required to develop strategies to enhance student support in academic practice.

Breast cancer is a leading cause of cancer-related deaths. Anemia is common in breast cancer patients and can be treated with blood transfusions or with recombinant erythropoietin (EPO) to stimulate red blood cell production. Clinical studies have indicated decreased survival in some groups of cancer patients treated with EPO. Numerous tumor cells express the EPO receptor (EPOR), posing a risk that EPO treatment would enhance tumor growth, but the mechanisms involved in breast tumor progression are poorly understood. Here, we have examined the functional role of the EPO-EPOR axis in pre-clinical models of breast cancer. EPO induced the activation of PI3K/AKT and MAPK pathways in human breast cancer cell lines. EPOR knockdown abrogated human tumor cell growth, induced apoptosis through Bim, reduced invasiveness, and caused downregulation of MYC expression. EPO-induced MYC expression is mediated through the PI3K/AKT and MAPK pathways, and overexpression of MYC partially rescued loss of cell proliferation caused by EPOR downregulation. In a xenotransplantation model, designed to simulate recombinant EPO therapy in breast cancer patients, knockdown of EPOR markedly reduced tumor growth. Thus, our experiments in vitro and in vivo demonstrate that functional EPOR signaling is essential for the tumor-promoting effects of EPO and underline the importance of the EPO-EPOR axis in breast tumor progression.

Development of anti-cancer drugs towards clinical application is costly and inefficient. Large screens of drugs, efficacious for non-cancer disease, are currently being used to identify candidates for repurposing based on their anti-cancer properties. Here, we show that low-dose salinomycin, a coccidiostat ionophore previously identified in a breast cancer screen, has anti-leukemic efficacy. AML and MLLr cell lines, primary cells and patient samples were sensitive to submicromolar salinomycin. Most strikingly, colony formation of normal hematopoietic cells was unaffected by salinomycin, demonstrating a lack of hemotoxicity at the effective concentrations. Furthermore, salinomycin treatment of primary cells resulted in loss of leukemia repopulation ability following transplantation, as demonstrated by extended recipient survival compared to controls. Bioinformatic analysis of a 17-gene signature identified and validated in primary MLLr cells, uncovered immunomodulatory pathways, hubs and protein interactions as potential transducers of low dose salinomycin treatment. Additionally, increased protein expression of p62/Sqstm1, encoded for by one of the 17 signature genes, demonstrates a role for salinomycin in aggresome/vesicle formation indicative of an autophagic response. Together, the data support the efficacy of salinomycin as an anti-leukemic at non-hemotoxic concentrations. Further investigation alone or in combination with other therapies is warranted for future clinical trial.

Inhibition of the chaperone heat-shock protein 90 (HSP90) induces apoptosis, and it is a promising anti-cancer strategy. The mechanisms underpinning apoptosis activation following HSP90 inhibition and how they are modified during acquired drug resistance are unknown. We show for the first time that, to induce apoptosis, HSP90 inhibition requires the cooperation of multi BH3-only proteins (BID, BIK, PUMA) and the reciprocal suppression of the pro-survival BCL-2 family member MCL1, which occurs via inhibition of STAT5A. A subset of tumour cell lines exhibit dependence on MCL1 expression for survival and this dependence is also associated with tumour response to HSP90 inhibition. In the acquired resistance setting, MCL1 suppression in response to HSP90 inhibitors is maintained; however, a switch in MCL1 dependence occurs. This can be exploited by the BH3 peptidomimetic ABT737, through non-BCL-2-dependent synthetic lethality.

Leukemia is the most frequently (30%) occurring type of pediatric cancer. Of these, approximately 80% are acute lymphoblastic leukemia (ALL) with acute myeloid leukemia (AML) cases making up the remaining 20%. Management of ALL has improved dramatically over the years with around 90% of patients surviving 5 years or more. Unfortunately, children with AML do not have such a promising prognosis with only 60% surviving 5 years or longer, plus a high risk of relapse, the side effects of the harsh chemotherapy regimen and a risk of not responding to induction therapy, provide a bleak outlook. Recently, Bolouri et al., (2017) identified the need for age-specific therapies for AML patients. To work towards age-specific treatment regimens we used the TARGET AML transcriptomic data, (https://ocg.cancer.gov/programs/target/data-matrix) and identified several therapeutically targetable pathways, in particular anomalies in apoptotic pathways amongst cytogenetically normal relapsed AML patients. Based on these apoptotic pathways we screened 80 apoptosis-inducing drugs from a compound library obtained from Selleckchem. The effect of three doses (0.01 µM, 0.1 µM and 1 µM) over 72 hrs of each drug was assayed using CellTox™ Green reagent (Promega) to investigate the cytotoxic potential of the compounds against three pediatric AML cell lines (Kasumi-1, MV4-11 and CMK) representative of a spectrum of cytogenetic and mutational anomalies. The cell lines showed varying responses in a time and dose-dependent manner with mutual and unique effects being highlighted, six compounds, including Navitoclax and TP-0903, overlapped in their effect on cell viability between the three cell lines. The cell lines also showed unique responses to many compounds, including Sunitinib and Obatoclax in the MV4-11 cell line. Based on these findings a dose of 0.1 µM was taken forward for the combination screen. To carry out the combination screen we used R to compile an algorithm, with the capability of determining all possible pairwise combinations for a given number of compounds. Several inputs were accommodated, including: the number of compounds to be used; the number of compounds in a well and the number of times the algorithm should run to get the best solution, with the best solution being the least number of wells to reduce redundant repeats of pairs. The output detailed which compounds should be in which well. With an input of 80 apoptotic-inducing compounds into the algorithm; the output was as follows: 3,160 possible pairwise combinations, 1,197 pairs used once, 827 pairs used twice, and so on with the largest number of repeats being 148 pairs used eight times. This output enabled us to cross validate our data, by analysing wells containing the successful combinations to corroborate them as 'hits' or as containing possible antagonistic pairings if they were deemed unsuccessful. Using the output of the algorithm, 160 wells were needed for all combinations to enable our Multiplex Screening for Interacting Compounds for pediatric AML (MuSICAL). CellTox™ Green was used to determine cytotoxicity up to 72 hours and the same three cell lines used as for the single agent screen. The MuSICAL screen identified some wells in common amongst the three cell lines (6.25%), although many of the positive wells (12.5%) were unique to each of the cell lines. Well 158, which included Nutlin-3, Embelin and Poziotinib, was common for the MV4-11 and the CMK cell lines, with the individual compounds in this well having no substantial effect on cell viability as single agents, but a combination of 2 or more compounds induced substantial cell death. The 10 drugs in well 158 were deconvoluted and screened, resulting in 45 pairwise combinations. Several pairwise combinations, at 0.1 µM each, induced cell death in the MV4-11 and CMK cell lines. The most effective pairwise combination was the combination of ABT-737, a Bcl-2 inhibitor and Purvalanol A, a CDK inhibitor, in the MV4-11 cell line. The effect of this combination was also a 'hit' in Well 6 validating the therapeutic potential. For the CMK cell line, the most effective combination was Poziotinib, a pan-HER inhibitor and SU9516, another CDK inhibitor. In conclusion, the MuSICAL screen has identified multiple apoptosis-inducing drug combinations across the 3 cell lines highlighting the potential for personalised treatment of pediatric AML with different cytogenetic and mutational backgrounds. Disclosures No relevant conflicts of interest to declare.

Background: Germline mutation screening of BRCA1 and BRCA2 genes is performed in suspected familial breast cancer cases, but a causative mutation is found in only 30% of patients. The development of additional methods to identify good candidates for BRCA1 and BRCA2 analysis would therefore increase the efficacy of diagnostic mutation screening. With this in mind, we developed a study to determine molecular signatures of BRCA1—or BRCA2—mutated breast cancers. Materials and Methods: Array-cgh (comparative genomic hybridization) and transcriptomic analysis were performed on a series of 103 familial breast cancers. The series included 7 breast cancers with a BRCA1 mutation and 5 breast cancers with a BRCA2 mutation. The remaining 91 cases were obtained from 73 families selected on the basis of at least 3 affected first-degree relatives or at least 2 affected first-degree relatives with breast cancer at an average age of 45 years. Array-cgh analyses were performed on a 4407 BAC-array (CIT-V8) manufactured by IntegraGen. Transcriptomic analyses were performed using an Affymetrix Human Genome U133 Plus 2.0 chip. Results: Using supervised clustering analyses we identified two transcriptomic signatures: one for BRCA1-mutated breast cancers consisting of 600 probe sets and another for BRCA2-mutated breast cancers also consisting of 600 probes sets. We also defined cgh-array signatures, based on the presence of specific genomic rearrangements, one for BRCA1-mutated breast cancers and one for BRCA2-mutated breast cancers. Conclusions: This study identified molecular signatures of breast cancers with BRCA1 or BRCA2 germline mutations. Genes present in these signatures could be exploited to find new markers for such breast cancers. We also identified specific genomic rearrangements in these breast cancers, which could be screened for in a diagnostic setting using fluorescence in situ hybridization, thus improving patient selection for BRCA1 and BRCA2 molecular genetic analysis.

Recombinant human erythropoietin (rHuEPO) is an effective treatment for anaemia but concerns that it causes disease progression in cancer patients by activation of EPO receptors (EPOR) in tumour tissue have been controversial and have restricted its clinical use. Initial clinical studies were flawed because they used polyclonal antibodies, later shown to lack specificity for EPOR. Moreover, multiple isoforms of EPOR caused by differential splicing have been reported in cancer cell lines at the mRNA level but investigations of these variants and their potential impact on tumour progression, have been hampered by lack of suitable antibodies. The EpoCan consortium seeks to promote improved pathological testing of EPOR, leading to safer clinical use of rHuEPO, by producing well characterized EPOR antibodies. Using novel genetic and traditional peptide immunization protocols, we have produced mouse and rat monoclonal antibodies, and show that several of these specifically recognize EPOR by Western blot, immunoprecipitation, immunofluorescence, flow cytometry and immunohistochemistry in cell lines and clinical material. Widespread availability of these antibodies should enable the research community to gain a better understanding of the role of EPOR in cancer, and eventually to distinguish patients who can be treated safely by rHuEPO from those at increased risk from treatment.

In 1953, noting a remarkable consistency between the agents causing mutations and those associated with cancer, Carl Nordling, a Finnish-born architect, proposed that cancer results from an accumulation of genetic mutations. It is now generally accepted that inherited mutations and environmental carcinogens can lead to the development of premalignant clones. After further mutations, one cell reaches a critical state which confers a survival or growth advantage over normal cells. Such cells have the ability to initiate a malignant tumour. They share many of the features of normal stem cells, including the capacity for self-renewal and differentiation, and are widely termed cancer stem cells (CSCs). Although CSCs have been well characterized in hematological malignancies, their existence in some other tissues has been questioned. Here, we review recent work in which stem cells and stem cell-like cells have been used to investigate the pathogenesis of cancer and potential anticancer treatment strategies, in the context of both hematological and somatic tissue disease. Stem Cells 2014;32:2563–2570

Ran is a small ras-related GTPase that controls the nucleocytoplasmic exchange of macromolecules across the nuclear envelope. It binds to chromatin early during nuclear formation and has important roles during the eukaryotic cell cycle, where it regulates mitotic spindle assembly, nuclear envelope formation and cell cycle checkpoint control. Like other GTPases, Ran relies on the cycling between GTP-bound and GDP-bound conformations to interact with effector proteins and regulate these processes. In nucleocytoplasmic transport, Ran shuttles across the nuclear envelope through nuclear pores. It is concentrated in the nucleus by an active import mechanism where it generates a high concentration of RanGTP by nucleotide exchange. It controls the assembly and disassembly of a range of complexes that are formed between Ran-binding proteins and cellular cargo to maintain rapid nuclear transport. Ran also has been identified as an essential protein in nuclear envelope formation in eukaryotes. This mechanism is dependent on importin-β, which regulates the assembly of further complexes important in this process, such as Nup107-Nup160. A strong body of evidence is emerging implicating Ran as a key protein in the metastatic progression of cancer. Ran is overexpressed in a range of tumors, such as breast and renal, and these perturbed levels are associated with local invasion, metastasis and reduced patient survival. Furthermore, tumors with oncogenic KRAS or PIK3CA mutations are addicted to Ran expression, which yields exciting future therapeutic opportunities.

A vast body of research in breast cancer prognostication has accumulated. Yet despite this, patients within current prognostic categories may have significantly different outcomes. There is a need to more accurately divide those cancer types associated with an excellent prognosis from those requiring more aggressive therapy. Gene expression array studies have revealed the numerous molecular breast cancer subtypes that are associated with differing outcomes. Furthermore, as next generation technologies evolve and further reveal the complexities of breast cancer, it is likely that existing prognostic approaches will become progressively refined. Future prognostication in breast cancer requires a morphomolecular, multifaceted approach involving the assessment of anatomical disease extent and levels of protein, DNA and RNA expression. One of the major challenges in prognostication will be the integration of potential assays into existing clinical systems and identification of appropriate patient subgroups for analysis.

Recombinant erythropoietin (Epo) is an effective anti-anemic agent in most cancer patients and improves their quality of life. Yet, concerns of disease progression and reduced survival in recombinant human Epo (rhuEpo)-treated patients have been raised by Phase III clinical trial data showing more rapid cancer progression and reduced survival in subjects randomized to Epo. Epo has pleiotropic actions and its receptor, EpoR, is expressed by many different cell types outside the erythroid compartment. It was thus proposed that a major possible mechanism for this potentially harmful effect of Epo in cancer patients is the activation of EpoRs on cancer cells. The original clinical studies have been criticized because they deployed polyclonal antibodies later shown to lack specificity for EpoR. Furthermore, multiple isoforms of EpoR caused by differential splicing have been reported, but only at the RNA level, in different cancer cell lines. Investigations into whether these spliced versions actually result in abnormal EpoR forms at the protein level, alter Epo responsiveness and have an impact on tumor progression in vivo, have been hampered by a lack of well characterized monoclonal antibodies. The ‘EpoCan’ Consortium, funded by the EU, is directed to promote improved pathological testing of EpoR in patient samples, leading to safer clinical use of rHuEpo and other Erythropoietic Stimulating Agents (ESAs). To date, 25 murine and rat monoclonal antibodies have been produced against the EpoR, using novel genetic and traditional peptide immunization protocols. Of these antibodies, several were found to specifically recognize the receptor in various assays including Western blot (WB), immunoprecipitation (IP), immunofluorescence (IF), flow cytometry (FACS) and immunohistochemistry (IHC). Table 1 lists the antibodies that were selected for further analysis, and their experimental applications. The antibodies were tested on EpoR transfected cells (HEK 293 cells and COS cells) and Epo-dependent UT7 cells which endogenously express EpoR. In addition the antibodies were tested on non-erythroid cells viz. the non-small cell lung carcinoma A549, and breast cancer MDA-MB 231 cell lines. Specificity of the antibodies towards the EpoR in these two latter cell lines was ensured by the lack of reactivity with the corresponding EpoR silenced cells.Table 1Anti EpoR Antibodies - immunizing antigens and applicationsImmunogenSubclone nameSystematic nameIsotypeEpitope locationApplicationsPeptideBCO-3H2-D3GM1201rIgG2bhEpoR cytoplasmic domainWB, IP, IF, IHCPeptideBCO-4B5-C9GM1202rIgG2ah/mEpoR cytoplasmic domainWB, IF, IHCDNAVP-2E8-B6GM1204mIgG1hEpoR extracellular domainIP, IF, FACSDNAVP-4D8-C4GM1205mIgG1hEpoR extracellular domainIP, IF, FACSDNABBQ-9C4-D8GM1206rIgG2ah/mEpoR extracellular domainFACSDNABBQ-10E10-F2GM1207rIgG2ah/mEpoR extracellular domainWB Immunohistochemical analysis was performed on a panel of human non-small cell lung carcinoma sections. Two rat monoclonal antibodies, BCO-3H2-D3 which recognizes full length EpoR and BCO-4B5-C9 which recognizes full length and truncated isoforms, have proved valuable in comparative immunohistochemical and Western blot studies in a range of human tumors and cell lines. These well characterized monoclonal antibodies will enable a careful dissection of EpoR function in cancer cells and the detection of EpoR isoforms in tumor tissue. In the longer term such studies should allow clinicians to balance the benefits and risks of ESA treatment in cancer. THIS WORK WAS SUPPORTED BY THE FP7-HEALTH EUROPEAN COMMISSION EPOCAN GRANT (282551). Disclosures: Grunert: Aldevron Freiburg GmBH: Employment. Thompson:Aldevron Freiburg GmbH: Employment.

Septins are members of a highly conserved family of 14 genes whose products are thought to act primarily as scaffolds, recruiting other proteins to particular cellular locations. This gene family shares a complexity of alternative splicing and encodes proteins involved in a number of diverse processes within the cell including cytokinesis, apoptosis and vesicle trafficking, and septins have been implicated in a number of diseases. Work in our laboratory has focussed on the SEPT9 gene and it was mapped it to a region of allelic imbalance on chromosome 17q in sporadic epithelial ovarian tumours. SEPT9 undergoes extensive alternative splicing and can form a possible 18 transcripts due to splicing at the 5’ (v1, v2, v3, v4, v4*, v5) and 3’ (a, b and c) ends, with SEPT9_v4 and SEPT9_v4* differing only in their 5’UTRs thus encoding the same protein. Previously we have shown perturbed expression of the SEPT9_v1 and SEPT9_v4* transcripts in neoplasia. In this study, we sought to determine the expression levels of all SEPT9 transcripts in ovarian tumours using samples from the Scottish Randomised Trial in Ovarian Cancer (SCOTROC) cohort, which compares two chemotherapy regimes. RNA was extracted from 100 FFPE tumour samples and qRT-PCR was performed for each SEPT9 transcript. Results were correlated with clinical data provided from the trial. Statistical significant elevated expression of SEPT9_v1 and SEPT9_v4* (p 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 5012. doi:10.1158/1538-7445.AM2011-5012

The septins are a family of 14 genes, the products of which are thought to function primarily as scaffolds which recruit other proteins to particular cellular locations. This gene family is highly conserved and encodes proteins involved in a variety of cellular functions including cytokinesis, apoptosis and vesicle trafficking. Septin colocalisation with actin and tubulin has also been reported. Previous work from this laboratory mapped the SEPT9 gene to a region of allelic imbalance on chromosome 17q in sporadic epithelial ovarian tumours. SEPT9 undergoes extensive alternate splicing at both the 5’ (v1, v2 v3, v4, v4* and v5) and 3’ (a, b and c) ends, with SEPT9_v4 and _v4* differing only in their 5’UTRs, therefore encoding the same polypeptide. We have shown perturbed expression of SEPT9 transcripts in neoplasia, specifically up-regulation of SEPT9_v1 and _v4* transcripts. In a range of normal tissues we noted three alternatively spliced 3’ transcripts (a, b & c) were expressed. However, in ovarian tumour development expression of 2 of these (b & c) is decreased. This suggests that expression of the various SEPT9 transcripts is independently regulated. We therefore sought to gain some insight into control of expression of these various splice variants and why specific patterns of deregulation might contribute to neoplasia. With respect to the cell cycle, we observed by immunofluoresence that SEPT9 protein is visible around the cell membrane during anaphase and into telophase. During cytokinesis the SEPT9 protein localises to the cleavage furrow and is part of the ‘ring’ structure of dividing cells. We also used a double thymidine block to synchronise HeLa cells but there was no alteration in levels of transcripts with specific 5’ or 3’ ends in these cells. Next we looked at expression of the SEPT9 5’ and 3’ ends in response to cellular stress, in particular DNA damage following doxorubicin treatment. We observe an up-regulation of the 5’ v4* transcript and 3’ c variant after doxorubicin treatment in a p53 wild type background but not in p53 null cells. We then used a model of ovarian surface epithelial cells immortalised with telomerase and a temperature sensitive SV40 large T antigen which proliferate at 330C but senesce when shifted to the non-permissive temperature (370C). We see that SEPT9_v2 and v4 mRNA levels increase significantly as cells undergo senescence. Finally, we note that the b and c variants share the same short 3’UTR (57 bases) while the a variant has a long 3’UTR (1.938kb). Bioinformatics of both 3’UTRs identified several putative microRNA binding sites present in the 3’UTR of the a variant but only one in that of b and c. We show that the 3’UTR of a drives a reporter gene when cloned upstream of luciferase and in a sequence dependant manner. In summary, our data supports the hypothesis that the alternatively spliced transcripts of SEPT9 play individual roles within the cell and that the 3’UTRs appear to be important in the regulation of SEPT9. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 260.