Lorne Cancer Conference 2003

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The Walter and Eliza Hall Institute of Medical Research, VIC


Cancer and programmed cell death: A report on the 15th Lorne Cancer Conference, 13–16 February 2003

The unifying theme of the 2003 Lorne cancer conference was set by Nobel Laureate, H Robert Horvitz (Howard Hughes Medical Institute and Massachusetts Institute of Technology, Cambridge, USA), in the plenary address describing his seminal work on the genetic control of programmed cell death in Caenorhabditis elegans. Apoptosis, as he pointed out, is a major feature of normal development, and disruption of this process results in a variety of human disorders, including cancer.

Cell death

The Nobel prize in physiology or medicine for 2002 was awarded to Sydney Brenner (The Salk Institute, La Jolla, USA), John Sulston (University of Cambridge, UK) and Horvitz for their combined work in establishing Caenorhabditis elegans as an experimental model organism and elucidating the genetic pathway for programmed cell death. Horvitz extended the work of Brenner and Sulston by identifying the central genes in the cell death (ced) pathway, including egl-1, ced-3, ced-4 and ced-9. Loss of function mutations in egl-1, ced-3 and ced-4 mutants cause the 131 cells normally fated to die by programmed cell death to survive in the adult hermaphrodite. Current work in the Horvitz lab centers on understanding the signals between cells undergoing programmed cell death and the cells that will engulf them. In a typically simple yet powerful screen, ced-3 hypomorphic mutant worms have been made transgenic for a reporter construct comprising green fluorescent protein (GFP) under the lin11 promoter (lin11::GFP), which marks ventral cord cells that are fated to undergo programmed cell death. A combination of mutagenesis and direct observation of GFP fluorescence in live worms has led to the identification of a number of mutations in known and novel genes that act in this pathway. Two mutations disrupting the engulfment process have been mapped in mutant strains, n3380 and n3376, identifying the dpl1 and MCD-1 genes respectively.

Two major pathways control the caspase-dependent apoptosis of a cell, namely the extrinsic and intrinsic pathways. The dogma in the field has suggested that initiation of the intrinsic pathway of apoptosis, which can be activated in response to cytotoxic stress such as DNA damage, requires permeabilization of the mitochondria and subsequent formation of the apoptosome, a protein complex made up of cytochrome c, Apaf-1 and caspase-9. Given that cytochrome c release in C elegans and Drosophila is not required for apoptosis, however, several groups have hypothesised that some apoptotic pathways are independent of the mitochondria and the apoptosome, and that the mitochondria serve to amplify but not to initiate the apoptotic caspase cascade.

Yuri Lazebnik (Cold Spring Harbor Laboratory, USA) described the use of the increasingly popular technology of small interfering RNA (siRNA) to inhibit the production of caspase-2. Using siRNA avoids the need to produce gene knockouts and does not appear to produce the anti-viral responses that are triggered by traditional antisense RNA methods. He and his coworkers have demonstrated that inhibition of caspase-2 production using siRNA can inhibit the translocation of the proapoptotic protein, Bax, from the cytoplasm to the mitochondria following induction of DNA damage with the drug etoposide. When translocated to the mitochondria, Bax is involved in mitochondrial permeabilization and release of cytochrome c. Thus, caspase-2 activation precedes activation of the well-characterized apoptosome cell death machinery. One obvious implication of this work is that a detailed study of caspase-2 activation in human tumours may provide insight into the failure of apoptosis in vivo and lead to the discovery of new therapeutic targets. Further evidence that caspase activation occurs upstream of the mitochondria and that the apoptosome functions to amplify the caspase cascade was presented by Vanessa Marsden (The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia). In order to study the requirement of Apaf-1 and caspase-9 in apoptosis, where Apaf-1 and caspase-9 deficiency is embryonic lethal, Marsden and colleagues reconstituted normal mice with foetal liver cells deficient in Apaf-1 and caspase-9. They found that lymphocyte apoptosis was largely unaffected by the loss of Apaf-1 and caspase-9 in response to growth factor withdrawal and stress.

One of the most interesting observations made by Lazebnik was that the field of apoptosis research is afflicted with an overwhelming productivity. This is manifest in the more than 10,000 papers that have been published yearly in this field for the past few years. How, Lazebnik asks, are we to assimilate and conceptualise all this information? To answer his own question, he created an analogy of a broken transistor radio. The functional properties of cellular signal transduction can be likened to those of a transistor radio; both are made up of many components which function together to receive, transduce and transmit specific signals. Could we as biologists, fix a broken radio? Firstly, we might obtain working examples of the radio and sooner or later find that we could remove the back to see inside. We would study it carefully and describe and categorise the size, morphology and colouring of all the components. Next, some scientists might begin a functional analysis by removing specific parts or cutting their connections to other parts. Finally we could arrive at a working model that might help us in diagnosing the problem with the original radio. If the radio is not working due to the lack of one component or a broken connection, or perhaps a fused and discolored component, no problem. We can replace the part and fix the radio. But what if the radio does not work as a result of a number of small changes in several tunable components not apparent in our analysis? We need an electrical engineer with a circuit diagram that describes the precise physical and functional relationships between the components that make up the transistor radio. Likewise, in order to understand cellular signal transduction, perhaps we need to develop a formal language to describe these relationships.

Cytokine signaling

In the signaling and cancer session, John O’Shea (National Institutes of Health, Maryland, USA) described finding patients with mutations in the cold autoinflammatory syndrome 1 (CIAS1) gene. Such mutations (of which 20 have been reported) result in a spectrum of diseases including neonatal-onset multisystem inflammatory disease (NOMID; also known as chronic infantile neurologic, cutaneous, articular, or CINCA, syndrome). O’Shea found that patients with CIAS1 mutations resulting in NOMID syndrome produce markedly elevated levels of IL-3, IL-5, IL-6, IL-1 and the IL-1 receptor antagonist, IL-1Ra. Interestingly, mutations in CIAS1, which encodes the protein cryopyrin, a regulator of NF-kB and the processing of IL-1, were found only in approximately 50% of the cases clinically identified as NOMID/CINCA syndrome. O’Shea proposed that heterogeneity in the promoter or intron regions of cryopyrin or cryopyrin homologs may explain the spectrum of diseases in humans with this genetic abnormality. Furthermore, he suggested that it may be possible to alleviate some of the symptoms of this disease using drugs that block IL-1 signaling.

Dendritic cells are specialised antigen presenting cells that coordinate immune responses to bacterial and viral pathogens. The development of dendritic cells is controlled both by interactions with the stromal environment, including T lymphocytes, and by cytokines, including those that signal through receptors bearing the signal-transducing gamma common (gamma c) chain, which is shared by receptors for IL-2, IL-4, IL-7, IL-9 and IL-15. Jak3 is an essential signalling component immediately downstream of the gamma c chain. Mice deficient in Jak3 are deficient in responses to IL-2, IL-4, IL-7 and IL-15. Morgan Wallace and collaborators have shown that Jak3 deficient mice display a three-fold decrease in CD8a+ splenic DC whereas CD11b+ splenic DC numbers were normal. CD11b+ cells are normally found in the marginal zone of the spleen and are responsible for Th2 T-helper cell responses, whereas CD8a+ dendritic cells are found in T-cell areas of the spleen and are probably the only dendritic cells capable of crosspriming CD8a+ T cells. To test whether the decrease in CD8a+ dendritic cells was a haematopoietic-specific defect, bone-marrow chimeras were generated using Jak3-deficient cells. The CD8a+ dendritic cells isolated from these chimeric mice showed a decrease in number and an increase in the expression of the CD40, B7.1 and B7.2 activation markers. Several possibilities have been suggested to account for the observed alterations in dendritic cell populations: activated Jak3-deficient T cells may kill CD8a+ dendritic cells or may provide inappropriate dendritic cell maturation signals; and disrupted splenic architecture may also prevent normal maturation. An alternative hypothesis revealed at the conference is that Jak3-deficient T cells produce a three- to five-fold increase in levels of IL-10, providing a possible negative feedback signal for CD8a+ dendritic cell development.

Cancer therapeutics

The conference included a number of notable presentations on current research into cancer therapeutics. The applications of combinatorial library technology were outlined by Kit Lam (University of California, Davis, USA) in a talk that focused on anti-cancer drug development and cancer proteomics. Combinatorial peptide chemistry is a blossoming field that is increasingly being utilised for the identification of cell-surface ligands and lymphocyte epitopes, and for studies of peptides binding to the major histocompatability complex (MHC), as well as vaccine development. The one-bead one-compound library method was first described by Lam in 1991: each 80-100µm bead expresses approximately 1013 copies of a unique peptide (of which there are up to 109 permutations). These peptide-coated beads are capable of binding a desired target, such as an antibody, protein, cell-surface receptor, tumour cell, virus or bacteria. Individual beads bearing a particular peptide can be isolated and sequenced by Edman degradation. This year, Lam described a peptide library screen designed to identify specific peptides that bind to tumour cells but not to normal cells. It is envisaged that these peptide agents could be used to target drugs to tumour cells. Tumour-specific peptides prepared using D-amino acids would be less likely to be rapidly degraded in vivo or to induce immune responses and might therefore be optimal as therapeutics.

The conference was the occasion of the inaugural Ashley Dunn oration. For the past decade, the Lorne Cancer Conference has been chaired by Ashley Dunn (Ludwig Institute for Cancer Research, Melbourne, Australia). The success of the conference during this time is undoubtedly a reflection of the patience, commitment and grace under pressure that has been a characteristic of Ashley’s tenure. The Ashley Dunn oration acknowledges the contribution Ashley has made not only to the Lorne Cancer Conference but to fostering research excellence within the Australian research community. Mary-Claire King (Department of Medicine and Genome Science, Seattle, USA) delivered the inaugural Ashley Dunn oration for 2003. The genetic analysis of breast and ovarian cancer, past, present and future was the subject of her lecture. The first BRCA1 mutations in families with inherited breast cancer were described by King and others in the early 1990s. They have continued to define the spectrum of mutations present in the BRCA1 and BRCA2 genes in familial breast and ovarian cancer and to search for other genes linked to this disease. Recently, King and co-workers observed that most of the BRCA1 mutations cause truncation and loss of the carboxy terminal transactivation domain. They have identified a number of direct transcriptional targets of BRCA1, including MYC and cyclinD1 which are frequently overexpressed in breast tumours.

In his concluding remarks, Douglas Hilton (The Walter and Eliza Hall Institute of Medical Research, Australia) noted that the 2003 Lorne Cancer Conference had brought together a group of leading scientists in a beautiful beachside location with anticipated results. Local and international speakers had outlined the central role of apoptosis and the cell death machinery in cancer. The importance of cytokine signalling regulation and cell cycle control in tumourigenesis was highlighted and several developments in tumour diagnosis and therapeutic strategies were revealed. In summary, the 15th annual Lorne conference confirmed that breakthroughs in the cancer problem can be achieved by utilising the power of diverse yet complementary approaches.

Acknowledgements

We acknowledge Douglas Hilton and Warren Alexander for their roles as joint chairmen of the Lorne Cancer Conference organising committee, and Helene Martin for her role as meeting co-ordinator.

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