The Walter and Eliza Hall Institute of Medical Research, VIC
Major themes presented at the 16th Lorne Cancer Conference included haematopoiesis, cell cycle, genomics and cancer therapy, for which a number of distinguished speakers were invited. Plenary orations this year were delivered by multiple award winner Janet Rowley (University of Chicago) and Nobel Laureate Lee Hartwell (Fred Hutchison Cancer Research Centre).
The conference began with a now traditional joint session with the preceding 29th Annual Conference on Protein Structure and Function. Steven Stacker (Ludwig Institute for Cancer Research, Melbourne, Australia) outlined the biochemistry of VEGF stimulation of lymphangiogenesis and how this may affect cancer metastasis and lymphedema. The session’s second speaker, David Carling (MRC Clinical Sciences Centre, London, UK) explained the importance of the AMP-regulated protein kinase in the regulation of cellular energy balance and how this relates to disease. Specifically, mutations in LKB1, a kinase upstream of AMPK has been shown to be responsible for inherited cancer (Pertz-Jeghers syndrome) and that the biochemical basis for this is the requirement of LKB1 for the function of AMPK.
As one of the major themes of the conference, cell cycle control was addressed by Patrick O’Farrell (University of California San Francisco, San Francisco, USA) as an incompletely explained phenomenon. In several elegant movies cell division was demonstrated to take far longer than is commonly perceived, as the cytoplasmic bridge that joins cells prior to division was observed to persist for hours after furrow ingression was completed. In addition, the textbook definition of cytokinesis was shown to be incomplete, in that chromosomal migration away from the kinetochore and toward the cell poles (typically after laser separation) does not occur appropriately outside its normal place in the cell cycle. To understand what facilitates this, a screen of RNAi mutants inducing multinucleation of drosophila cells was investigated. Fascinating mutants were described including knockouts for Rho-dependant kinases Citron kinase and Rock. Ablation of Rock stopped anaphase B elongation of the cell and meant that the spindle collided with the poles of the cell and that the spindle fibres buckled drastically towards the cell equator. Citron Kinase was shown to be involved in stabilisation of the cytoplasmic bridge, as its absence meant the bridge was not broken, and cells abruptly fused (in about one minute) nearly two hours after furrow ingression occurred.
Also utilising the well-described physiology of Drosophila melanogaster, Iswar Hariharan (MGH Center for Cancer Research, Charlestown, USA) conducted a screen for mutations affecting cell growth by comparing proliferation of wildtype and mutant cells in the fly eye on the basis of pigmentation. In this fashion several unpublished negative regulators of cell growth were identified. The F-box protein Archipelago was shown to degrade both myc and Cyclin E, thus affecting cell growth. A novel kinase complex affecting cell growth was also defined, comprising two protein kinases (Hippo and Warts) scaffolded by a third protein (Salvador). This complex restricts cell growth and promotes apoptosis making it a potentially efficacious tumour suppressor. Lastly, unpublished data concerning Capicua (Cic) was presented, identifying its role in growth and the receptor tyrosine kinase / Ras pathway. Cic mutant cells grow faster but with normal size and specification as opposed to other mutants downstream of Ras, for example Pointed mutants, which fail to specify photoreceptor cells. This leads to the interesting hypothesis that cellular signals for growth and specification downstream of Ras are independently controlled.
Predisposition to cancer is one of the potential results of defects in DNA damage responses. Kum Kum Khanna (Queensland Institute of Medical Research, Brisbane, Australia) highlighted that these defects can also lead to immune deficiencies, neurodegeneration, telomere defects and ageing, thus, her work focuses on the stability of DNA and cell cycle checkpoints, particularly in response to DNA damage. ATM (ataxia-telangiectasia mutated) is an important protein kinase involved in the regulation of checkpoint responses and is recruited to sites of double-stranded breaks of DNA. Mice deficient in ATM are sensitive to ionising radiation, lose G1, S and G2/M checkpoints and demonstrate chromosome instability.
Also researching the cell cycle Bruce Stillman (Cold Spring Harbor Laboratory, New York, USA) explained work on Origin Recognition Complex (ORC) proteins which are involved in marking sites of DNA for replication. Orc2 was knocked down in eukaryotic cells using siRNA and led to lack of DNA replication and arrest in mitotic state associated with severe spindle defects. Protein staining of these cells suggested an altered composition of the centrosome and analysis of spindle checkpoint proteins denoted an absence of Mad2 on the kinetochores, raising issues about Mad2-dependent checkpoint signalling in mitosis. As presented at this conference, some of the complexities underlying the association of cell cycle and cancer are being elucidated, exposing potential targets and pathways for future therapies
A week after celebrating 50 years in science at the Walter and Eliza Hall Institute of Medical Research (WEHI) Donald Metcalf (WEHI, Melbourne, Australia) was describing the phenotypes of two lines of mice (Plt3 and 4) arising from an ENU mutagenesis screen directed towards genes involved in the regulation of platelet production. These mice demonstrated mild anaemia, deficient levels of B lymphocytes and an increased level of megakaryocyte progenitors and platelets. The genotype of these mice showed that two independent point mutations in the myb gene lead to this phenotype. Interestingly, this is in contrast to the myb knockout mouse, which is not viable. The specific interactions abrogated by these point mutations should provide insight into important haematopoietic pathways relevant to platelet production.
Thea Tlsty (University of California San Francisco, San Francisco, USA) discussed her investigations of the early molecular and genetic events of breast cancer, a disease which is frequently diagnosed after appropriate treatment can be applied. She has developed an elegant in vitro model based on normal breast tissue which identifies signalling pathways and genes that enable cells to bypass normal proliferation barriers and acquire the genomic changes that are pivotal to the development of cancer. Amongst the molecular descriptions, increasing COX-2 expression appears to correlate with this breast cancer model and is overexpressed in many other solid tumours. Colocalisation of COX-2 and p16 (a molecule involved in cell cycle control) was also observed in a percentage of tumours studied. Currently, COX-2 inhibitors are prescribed for arthritis and inflammation but clinical trials are taking place to test their efficacy in the treatment of colon cancer.
Concerning the role of haematopoietic stem cells (HSCs) as a potential therapeutic agent through transplantation, Margaret Goodell (Baylor college of Medicine, Houston, USA) presented a large body of work towards an understanding of the nature of HSC differentiation and self-renewal upon transplantation. As potential proof of transdifferentiation, a single bone marrow derived HSC can proliferate in damaged liver. The process by which this occurs was not due to a switch in the fate of the cell, but rather engraftment, a rare process whereby fusion occurs of the transplanted HSC with a hepatocyte. This raised the point that hepatocytes by their nature being more accepting of a multinuclear, multiploid state, may show a preference for the process of transdifferentiation of HSCs. All in all the many facets of haematopoiesis relevant to cancer and its therapy highlighted its utility and importance to the conference.
Several speakers outlined recent innovations in research relevant to cancer. Michael Gould (McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, USA) has developed a yeast-based screen to analyse ENU mutaganised rats, and hence found those with loss of function for specific genes. Screening less than 2000 mutants Gould identified BRCA2 knockout rats, which unlike their mouse knockout counterparts, were viable. The BRCA2 knockout rat instead had a decreased life span, tumours, cataracts, kidney disease and an overt loss of sperm in the testes and germ cells from ovaries.
Also utilising ENU mutagenesis technology, this time in mice, Ben Kile (Baylor College of Medicine, Houston, USA) described a screen to bypass traditional bottlenecks in mapping and maintenance of ENU mutagenised mice. To do this a balancer chromosome was designed and used in a recessive mutant screen. 230 new recessive mutations were identified, 88 of which mapped alongside the balancer chromosome therefore enabling their rapid identification.
While investigation of protein networks is currently the focus of much attention, the generation of genetic interaction maps is not as prevalent. Brenda Andrews (Department of Medical Genetics and Microbiology, University of Toronto, Canada) has developed a method defining genetic interaction maps with up to 4000 interactions. This method is termed Synthetic Genetic Array (SGA) and involves analysis of yeast double mutants by crossing gene mutations into an array of viable mutants. Double mutants are then tested for phenotypes affecting viability and fitness which would place the two genes in associated pathways. Results suggested that the genetic interaction map is in the order of 100,000 interactions – possibly 10 times more complex than the protein interaction map. Furthermore, Andrews suggested that the position of a gene in a partially-mapped network was predictive of its interactions, in that a gene is more likely to be associated with the neighbours of a gene it already interacts with.
The Ashley Dunn Oration was delivered this year by Lee Hartwell (Fred Hutchison Cancer Research Center, Seattle, USA) who won the Nobel Prize for Medicine (physiology) in 2001. Hartwell initially provided an insightful review of cancer therapy, emphasising that the death rate from cancer over the last five decades has remained unchanged and that the number of drugs in the pipeline towards therapy is in decline. In what was clearly the most controversial proposition presented at the conference, Hartwell suggested that in fact the current dogma of cancer research is fundamentally flawed. Instead, as opposed to researching therapy, data presented showed that the most benefit science could deliver to cancer patients would be through early diagnosis by which recovery rates are drastically improved. The gauntlet was thrown down to researchers to drop therapeutic research in favour of diagnostic research, for which many tools are now becoming available. Hartwell envisages a day in which a handful (a ball-park figure of 100 was suggested) markers in serum could diagnose cancer specifically and lead to more efficacious treatment. The challenge will be to convince people with money to support such a vision, however data presented in this oration suggested that future victims of cancer would not be hard to convince.
The question of whether we are focusing on the right targets and strategies for combating cancer was also discussed by Janet Rowley (University of Chicago, Chicago, USA) in a plenary session on Haematopoiesis. Rowley has performed extensive research on the Philadelphia chromosome – from its discovery to cloning and its role in acute myeloid leukaemia. As cancers have distinct morphological features based on their genotype, concentrating on one gene at a time is less profitable than research using a more global approach. The Rowley lab has developed SAGE (serial analysis of gene expression) as a tool to analyse gene transcripts in an unbiased, comprehensive fashion. Data provided indicated that transcriptional activation in the genome is probably a magnitude higher than is currently believed. Therefore the conclusion to be drawn was that, especially in the treatment of cancer where mutations, translocation and epigenetics also play a role, genotypic-specific therapies will probably be the future of cancer research.
Although there is a continuing push for new and better therapies, the success of current therapeutics and clinical trials cannot be ignored. Many different therapies are being tested with positive results from clinical trials. These include using anti-VEGF antibodies (Avastin) which reduces tumor vascularisation hence apprehending tumour growth (Jennifer LeCouter, Genentech Inc, San Francisco, USA) and the development of a vaccine against human papilloma virus (Suzanne Garland, Royal Women’s Hospital, Melbourne, Australia) to decrease the risk of cervical cancer through persistent oncogenic HPV infection. The traditional treatments of chemotherapy and radiotherapy are often debilitating and induce complications including oral mucositis. Clinical trials are underway in the use of rhuKGF to induce epithelial cell proliferation and survival to decrease the severity and duration of this complication (Alessandra Cesano, AMGEN Inc, Thousand Oaks, California, USA).
Poster presentations this year were of uniformly outstanding quality and deserving of the six independently funded poster prizes. Marnie Champ was recognised for her contribution as the best postdoc/student speaker of the conference. In closing Doug Hilton (WEHI, Melbourne, Australia) highlighted three facets of the meeting which he believed were instrumental to its success. These were, the demonstration of stellar science by all contributing members, free student registration (provided by the anti-cancer council) and a social environment that contributes to new and continued friendships amongst the scientific community.
The authors acknowledge Doug Hilton and Warren Alexander, joint chairmen of the Lorne Cancer Conference Organising Committee, and also Helene Martin and Jacqui Laird, meeting coordinators.