Bodo Lange

Hsp90 is a core centrosomal component and is required at different stages of the centrosome cycle in Drosophila and vertebrates

To determine the molecular composition of the centrosome of a higher eukaryote, we carried out a systematic nano‐electrospray tandem or MALDI mass spectrometry analysis of the polypeptides present in highly enriched preparations of immunoisolated Drosophila centrosomes. One of the proteins identified is Hsp83, a member of the highly conserved Hsp90 family including chaperones known to maintain the activity of many proteins but suspected to have other essential, unidentified functions. We have found that a fraction of the total Hsp90 pool is localized at the centrosome throughout the cell cycle at different stages of development in Drosophila and vertebrates. This association between Hsp90 and the centrosome can be observed in purified centrosomes and after treatment with microtubule depolymerizing drugs, two criteria normally used to define core centrosomal components. Disruption of Hsp90 function by mutations in the Drosophila gene or treatment of mammalian cells with the Hsp90 inhibitor geldanamycin, results in abnormal centrosome separation and maturation, aberrant spindles and impaired chromosome segregation. This suggests that another role of Hsp90 might be to ensure proper centrosome function.

Asterless is a centriolar protein required for centrosome function and embryo development in Drosophila

BACKGROUND Centrosomes, the major organizers of the microtubule network in most animal cells, are composed of centrioles embedded in a web of pericentriolar material (PCM). Recruitment and stabilization of PCM on the centrosome is a centriole-dependent function. Compared to the considerable number of PCM proteins known, the molecular characterization of centrioles is still very limited. Only a few centriolar proteins have been identified so far in Drosophila, most related to centriole duplication. RESULTS We have cloned asterless (asl) and found that it encodes a 120 kD highly coiled-coil protein that is a constitutive pancentriolar and basal body component. Loss of asl function impedes the stabilization/maintenance of PCM at the centrosome. In embryos deficient for Asl, development is arrested right after fertilization. Asl shares significant homology with Cep 152, a protein described as a component of the human centrosome for which no functional data is yet available. CONCLUSIONS The cloning of asl offers new insight into the molecular composition of Drosophila centrioles and a possible model for the role of its human homolog. In addition, the phenotype of asl-deficient flies reveals that a functional centrosome is required for Drosophila embryo development.

Miniaturization in functional genomics and proteomics

Proteins are the key components of the cellular machinery responsible for processing changes that are ordered by genomic information. Analysis of most human proteins and nucleic acids is important in order to decode the complex networks that are likely to underlie many common diseases. Significant improvements in current technology are also required to dissect the regulatory processes in high-throughtput and with low cost. Miniaturization of biological assays is an important prerequisite to achieve these goals in the near future.

Proteomic and functional analysis of the mitotic Drosophila centrosome.

Regulation of centrosome structure, duplication and segregation is integrated into cellular pathways that control cell cycle progression and growth. As part of these pathways, numerous proteins with well‐established non‐centrosomal localization and function associate with the centrosome to fulfill regulatory functions. In turn, classical centrosomal components take up functional and structural roles as part of other cellular organelles and compartments. Thus, although a comprehensive inventory of centrosome components is missing, emerging evidence indicates that its molecular composition reflects the complexity of its functions. We analysed the Drosophila embryonic centrosomal proteome using immunoisolation in combination with mass spectrometry. The 251 identified components were functionally characterized by RNA interference. Among those, a core group of 11 proteins was critical for centrosome structure maintenance. Depletion of any of these proteins in Drosophila SL2 cells resulted in centrosome disintegration, revealing a molecular dependency of centrosome structure on components of the protein translation machinery, actin‐ and RNA‐binding proteins. In total, we assigned novel centrosome‐related functions to 24 proteins and confirmed 13 of these in human cells.

Cdc37 is essential for chromosome segregation and cytokinesis in higher eukaryotes.

Cdc37 has been shown to be required for the activity and stability of protein kinases that regulate different stages of cell cycle progression. However, little is known so far regarding interactions of Cdc37 with kinases that play a role in cell division. Here we show that the loss of function of Cdc37 in Drosophila leads to defects in mitosis and male meiosis, and that these phenotypes closely resemble those brought about by the inactivation of Aurora B. We provide evidence that Aurora B interacts with and requires the Cdc37/Hsp90 complex for its stability. We conclude that the Cdc37/Hsp90 complex modulates the function of Aurora B and that most of the phenotypes brought about by the loss of Cdc37 function can be explained by the inactivation of this kinase. These observations substantiate the role of Cdc37 as an upstream regulatory element of key cell cycle kinases.

Molecular dissection of colorectal cancer in pre-clinical models identifies biomarkers predicting sensitivity to EGFR inhibitors

Colorectal carcinoma represents a heterogeneous entity, with only a fraction of the tumours responding to available therapies, requiring a better molecular understanding of the disease in precision oncology. To address this challenge, the OncoTrack consortium recruited 106 CRC patients (stages I–IV) and developed a pre-clinical platform generating a compendium of drug sensitivity data totalling >4,000 assays testing 16 clinical drugs on patient-derived in vivo and in vitro models. This large biobank of 106 tumours, 35 organoids and 59 xenografts, with extensive omics data comparing donor tumours and derived models provides a resource for advancing our understanding of CRC. Models recapitulate many of the genetic and transcriptomic features of the donors, but defined less complex molecular sub-groups because of the loss of human stroma. Linking molecular profiles with drug sensitivity patterns identifies novel biomarkers, including a signature outperforming RAS/RAF mutations in predicting sensitivity to the EGFR inhibitor cetuximab.

The structural impact of cancer-associated missense mutations in oncogenes and tumor suppressors

BackgroundCurrent large-scale cancer sequencing projects have identified large numbers of somatic mutations covering an increasing number of different cancer tissues and patients. However, the characterization of these mutations at the structural and functional level remains a challenge.ResultsWe present results from an analysis of the structural impact of frequent missense cancer mutations using an automated method. We find that inactivation of tumor suppressors in cancer correlates frequently with destabilizing mutations preferably in the core of the protein, while enhanced activity of oncogenes is often linked to specific mutations at functional sites. Furthermore, our results show that this alteration of oncogenic activity is often associated with mutations at ATP or GTP binding sites.ConclusionsWith our findings we can confirm and statistically validate the hypotheses for the gain-of-function and loss-of-function mechanisms of oncogenes and tumor suppressors, respectively. We show that the distinct mutational patterns can potentially be used to pre-classify newly identified cancer-associated genes with yet unknown function.

LGALS3BP regulates centriole biogenesis and centrosome hypertrophy in cancer cells

Centrosome morphology and number are frequently deregulated in cancer cells. Here, to identify factors that are functionally relevant for centrosome abnormalities in cancer cells, we established a protein-interaction network around 23 centrosomal and cell-cycle regulatory proteins, selecting the interacting proteins that are deregulated in cancer for further studies. One of these components, LGALS3BP, is a centriole- and basal body-associated protein with a dual role, triggering centrosome hypertrophy when overexpressed and causing accumulation of centriolar substructures when downregulated. The cancer cell line SK-BR-3 that overexpresses LGALS3BP exhibits hypertrophic centrosomes, whereas in seminoma tissues with low expression of LGALS3BP, supernumerary centriole-like structures are present. Centrosome hypertrophy is reversed by depleting LGALS3BP in cells endogenously overexpressing this protein, supporting a direct role in centrosome aberration. We propose that LGALS3BP suppresses assembly of centriolar substructures, and when depleted, causes accumulation of centriolar complexes comprising CPAP, acetylated tubulin and centrin.

Personalized medicine approaches for colon cancer driven by genomics and systems biology: OncoTrack.

The post‐genomic era promises to pave the way to a personalized understanding of disease processes, with technological and analytical advances helping to solve some of the worlds health challenges. Despite extraordinary progress in our understanding of cancer pathogenesis, the disease remains one of the worlds major medical problems. New therapies and diagnostic procedures to guide their clinical application are urgently required. OncoTrack, a consortium between industry and academia, supported by the Innovative Medicines Initiative, signifies a new era in personalized medicine, which synthesizes current technological advances in omics techniques, systems biology approaches, and mathematical modeling. A truly personalized molecular imprint of the tumor micro‐environment and subsequent diagnostic and therapeutic insight is gained, with the ultimate goal of matching the “right” patient to the “right” drug and identifying predictive biomarkers for clinical application. This comprehensive mapping of the colon cancer molecular landscape in tandem with crucial, clinical functional annotation for systems biology analysis provides unprecedented insight and predictive power for colon cancer management. Overall, we show that major biotechnological developments in tandem with changes in clinical thinking have laid the foundations for the OncoTrack approach and the future clinical application of a truly personalized approach to colon cancer theranostics.

Health technology assessment in the era of personalized health care

OBJECTIVES This article examines the challenges for health technology assessment (HTA) in the light of new developments of personalized health care, focusing on European HTA perspectives. METHODS Using the example of the Integrated Genome Research Network - Mutanom (IG Mutanom) project, with focus on personalized cancer diagnostics and treatment, we assess the scope of current HTA and examine it prospectively in the context of the translation of basic and clinical research into public health genomics and personalized health care. RESULTS The approaches developed within the IG-Mutanom project are based on innovative technology potentially providing targeted therapies for cancer; making translation into clinical practice requires a novel course of action, however. New models of HTA are needed that can account for the unique types of evidence inherent to individualized targeted therapies. Using constructive health technology assessment (CTA) models is an option, but further suitable models should be developed. CONCLUSIONS Integrative, systems biology-based approaches toward personalized medicine call for novel assessment methods. The translation of their highly innovative technologies into the practice of health care requires the development of new HTA concepts.