Aaron J. Urquhart

Functions and therapeutic roles of exosomes in cancer.

The role of exosomes in cancer development has become the focus of much research, due to the many emerging roles possessed by exosomes. These micro-vesicles that are ubiquitously released in to the extracellular milieu, have been found to regulate immune system function, particularly in tumorigenesis, as well as conditioning future metastatic sites for the attachment and growth of tumor tissue. Through an interaction with a range of host tissue, exosomes are able to generate a pro-tumor environment that is essential for carcinogenesis. Herein, we discuss the contents of exosomes and their contribution to tumorigenesis, as well as their role in chemotherapeutic resistance and the development of novel cancer treatments and the identification of cancer biomarkers.

hSSB1 interacts directly with the MRN complex stimulating its recruitment to DNA double-strand breaks and its endo-nuclease activity

hSSB1 is a recently discovered single-stranded DNA binding protein that is essential for efficient repair of DNA double-strand breaks (DSBs) by the homologous recombination pathway. hSSB1 is required for the efficient recruitment of the MRN complex to sites of DSBs and for the efficient initiation of ATM dependent signalling. Here we explore the interplay between hSSB1 and MRN. We demonstrate that hSSB1 binds directly to NBS1, a component of the MRN complex, in a DNA damage independent manner. Consistent with the direct interaction, we observe that hSSB1 greatly stimulates the endo-nuclease activity of the MRN complex, a process that requires the C-terminal tail of hSSB1. Interestingly, analysis of two point mutations in NBS1, associated with Nijmegen breakage syndrome, revealed weaker binding to hSSB1, suggesting a possible disease mechanism.

ATM mediated phosphorylation of CHD4 contributes to genome maintenance

BackgroundIn order to maintain cellular viability and genetic integrity cells must respond quickly following the induction of cytotoxic double strand DNA breaks (DSB). This response requires a number of processes including stabilisation of the DSB, signalling of the break and repair. It is becoming increasingly apparent that one key step in this process is chromatin remodelling.ResultsHere we describe the chromodomain helicase DNA-binding protein (CHD4) as a target of ATM kinase. We show that ionising radiation (IR)-induced phosphorylation of CHD4 affects its intranuclear organization resulting in increased chromatin binding/retention. We also show assembly of phosphorylated CHD4 foci at sites of DNA damage, which might be required to fulfil its function in the regulation of DNA repair. Consistent with this, cells overexpressing a phospho-mutant version of CHD4 that cannot be phosphorylated by ATM fail to show enhanced chromatin retention after DSBs and display high rates of spontaneous damage.ConclusionThese results provide insight into how CHD4 phosphorylation might be required to remodel chromatin around DNA breaks allowing efficient DNA repair to occur.

Centrobin regulates the assembly of functional mitotic spindles

The proper function of the spindle is crucial to the high fidelity of chromosome segregation and is indispensable for tumor suppression in humans. Centrobin is a recently identified centrosomal protein that has a role in stabilizing the microtubule structure. Here we functionally characterize the defects in centrosome integrity and spindle assembly in Centrobin-depleted cells. Centrobin-depleted cells show a range of spindle abnormalities including unfocused poles that are not associated with centrosomes, S-shaped spindles and mini spindles. These cells undergo mitotic arrest and subsequently often die by apoptosis, as determined by live cell imaging. Co-depletion of Mad2 relieves the mitotic arrest, indicating that cells arrest due to a failure to silence the spindle checkpoint in metaphase. Consistent with this, Centrobin-depleted metaphase cells stained positive for BubR1 and BubR1 S676. Staining with a panel of centrosome markers showed a loss of centrosome anchoring to the mitotic spindle. Furthermore, these cells show less cold-stable microtubules and a shorter distance between kinetochore pairs. These results show a requirement of Centrobin in maintaining centrosome integrity, which in turn promotes anchoring of mitotic spindle to the centrosomes. Furthermore, this anchoring is required for the stability of microtubule–kinetochore attachments and biogenesis of tension-ridden and properly functioning mitotic spindle.

74P Elucidating drug resistance mechanisms using 2D and 3D culture systems.

targeted therapies. EGFR is commonly expressed in LSCC, while FGFR1 amplification and DDR2 mutations are less common. Necitumumab, an anti-EGFR monoclonal antibody (mAb), is the only targeted therapy approved, in combination with chemotherapy, as a 1st-line treatment of metastatic LSCC patients. A major clinical challenge is that all patients who initially benefit from targeted therapies eventually develop resistance. AXL overexpression, STAT3 or YAP pathway activation, as well as hedgehog-GLI signaling have been described as mechanisms of resistance to anti-EGFR therapy. p21-activated kinase 1 (PAK1), which regulates ERK, is overexpressed in LSCC, diminishing the efficacy of anti-EGFR mAbs. Herein, we are evaluating the relevance of these pathways in LSCC cell lines, illustrating the potential for synthetic lethal approaches. Methods: mRNA expression of AXL, STAT3, YAP, GLI2 and PAK1 were examined by quantitative real-time PCR in 6 LSCC cell lines: H2286, HCC366, H520, H1703, SK-MES-1, and EBC1. Results: EGFR was homogeneously overexpressed in all tested cell lines, but a differential expression pattern of the other transcripts was identified. Regarding the two DDR2 mutant cell lines, the H2286 had AXL and YAP overexpression, while the HCC366 had STAT3 overexpression. The analysis of the two FGFR1 amplified cell lines demonstrated that the H1703 had STAT3 overexpression, while the H520 cells overexpressed GLI2 and PAK1. High expression of PAK1 was also detected in the p53 mutant EBC-1 cell line. The results are the mean of three independent experiments. Immunoblotting analysis is ongoing while the combined effect of necitumumab with different drugs (STAT3, PAK1, GLI and AXL inhibitors), will also be evaluated. Conclusions: Diverse biological behavior of LSCC cancer cell lines may result from diverse underlying genetic profiles and gene expression. The awareness of these differences makes it necessary to take them into account, while planning efficient targeted strategies and synergistic drug interactions. Legal entity responsible for the study: Catalan Institute of Oncology Germans Trias i Pujol Health Sciences Institute and Hospital Campus Can Ruti 08916 Badalona, Barcelona Spain Funding: This Research Project was supported by ESMO with the aid of a grant from Roche. Disclosure: All authors have declared no conflicts of interest.