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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.

Breast cancer is one of the most prevalent malignancies worldwide. It consists of a group of tumor cells that have the ability to grow uncontrollably, overcome replicative senescence (tumor progression) and metastasize within the body. Metastases are processes that consist of an array of complex gene dysregulation events. Although these processes are still not fully understood, the dysregulation of a number of key proteins must take place if the tumor cells are to disseminate and metastasize. It is now widely accepted that future effective and innovative treatments of cancer metastasis will have to encompass all the major components of malignant transformation. For this reason, much research is now being carried out into the mechanisms that govern the malignant transformation processes. Recent research has identified key genes involved in the development of metastases, as well as their mechanisms of action. A detailed understanding of the encoded proteins and their interrelationship generates the possibility of developing novel therapeutic approaches. This review will focus on a select group of proteins, often deregulated in breast cancer metastasis, which have shown therapeutic promise, notably, EMT, E-cadherin, Osteopontin, PEA3, Transforming Growth Factor Beta (TGF-β) and Ran.

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.

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.

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.