Claudio R. Thoma

Global Identification of Modular Cullin-RING Ligase Substrates

Cullin-RING ligases (CRLs) represent the largest E3 ubiquitin ligase family in eukaryotes, and the identification of their substrates is critical to understanding regulation of the proteome. Using genetic and pharmacologic Cullin inactivation coupled with genetic (GPS) and proteomic (QUAINT) assays, we have identified hundreds of proteins whose stabilities or ubiquitylation status are regulated by CRLs. Together, these approaches yielded many known CRL substrates as well as a multitude of previously unknown putative substrates. We demonstrate that one substrate, NUSAP1, is an SCF(Cyclin F) substrate during S and G2 phases of the cell cycle and is also degraded in response to DNA damage. This collection of regulated substrates is highly enriched for nodes in protein interaction networks, representing critical connections between regulatory pathways. This demonstrates the broad role of CRL ubiquitylation in all aspects of cellular biology and provides a set of proteins likely to be key indicators of cellular physiology.

pVHL and GSK3|[beta]| are components of a primary cilium-maintenance signalling network

Defects in the structure or function of the primary cilium, an antennae-like structure whose functional integrity has been linked to the suppression of uncontrolled kidney epithelial cell proliferation, are a common feature of genetic disorders characterized by kidney cysts. However, the mechanisms by which primary cilia are maintained remain poorly defined. von Hippel-Lindau (VHL) disease is characterized by the development of premalignant renal cysts and arises because of functional inactivation of the VHL tumour suppressor gene product, pVHL. Here, we show that pVHL and glycogen synthase kinase (GSK)3β are key components of an interlinked signalling pathway that maintains the primary cilium. Although inactivation of either pVHL or GSK3β alone did not affect cilia maintenance, their combined inactivation leads to loss of cilia. In VHL patients, GSK3β is subjected to inhibitory phosphorylation in renal cysts, but not in early VHL mutant lesions, and these cysts exhibit reduced frequencies of primary cilia. We propose that pVHL and GSK3β function together in a ciliary-maintenance signalling network, disruption of which enhances the vulnerability of cells to lose their cilia, thereby promoting cyst formation.

3D cell culture systems modeling tumor growth determinants in cancer target discovery

Phenotypic heterogeneity of cancer cells, cell biological context, heterotypic crosstalk and the microenvironment are key determinants of the multistep process of tumor development. They sign responsible, to a significant extent, for the limited response and resistance of cancer cells to molecular-targeted therapies. Better functional knowledge of the complex intra- and intercellular signaling circuits underlying communication between the different cell types populating a tumor tissue and of the systemic and local factors that shape the tumor microenvironment is therefore imperative. Sophisticated 3D multicellular tumor spheroid (MCTS) systems provide an emerging tool to model the phenotypic and cellular heterogeneity as well as microenvironmental aspects of in vivo tumor growth. In this review we discuss the cellular, chemical and physical factors contributing to zonation and cellular crosstalk within tumor masses. On this basis, we further describe 3D cell culture technologies for growth of MCTS as advanced tools for exploring molecular tumor growth determinants and facilitating drug discovery efforts. We conclude with a synopsis on technological aspects for on-line analysis and post-processing of 3D MCTS models.

Analysis of microtubule dynamic instability using a plus-end growth marker

Regulation of microtubule dynamics is essential for many cell biological processes and is likely to be variable between different subcellular regions. We describe a computational approach to analyze microtubule dynamics by detecting growing microtubule plus ends. Our algorithm tracked all EB1-EGFP comets visible in an image time-lapse sequence allowing the detection of spatial patterns of microtubule dynamics. We introduce spatiotemporal clustering of EB1-EGFP growth tracks to infer microtubule behaviors during phases of pause and shortening. We validated the algorithm by comparing the results to data for manually tracked, homogeneously labeled microtubules and by analyzing the effects of well-characterized inhibitors of microtubule polymerization dynamics. We used our method to analyze spatial variations of intracellular microtubule dynamics in migrating epithelial cells.

pVHL and PTEN tumour suppressor proteins cooperatively suppress kidney cyst formation

In patients with von Hippel–Lindau (VHL) disease, renal cysts and clear cell renal cell carcinoma (ccRCC) arise from renal tubular epithelial cells containing biallelic inactivation of the VHL tumour suppressor gene. However, it is presumed that formation of renal cysts and their conversion to ccRCC involve additional genetic changes at other loci. Here, we show that cystic lesions in the kidneys of patients with VHL disease also demonstrate activation of the phosphatidylinositol‐3‐kinase (PI3K) pathway. Strikingly, combined conditional inactivation of Vhlh and the Pten tumour suppressor gene, which normally antagonises PI3K signalling, in the mouse kidney, elicits cyst formation after short latency, whereas inactivation of either tumour suppressor gene alone failed to produce such a phenotype. Interestingly, cells lining these cysts frequently lack a primary cilium, a microtubule‐based cellular antenna important for suppression of uncontrolled kidney epithelial cell proliferation and cyst formation. Our results support a model in which the PTEN tumour suppressor protein cooperates with pVHL to suppress cyst development in the kidney.

VHL loss causes spindle misorientation and chromosome instability

Error-free mitosis depends on fidelity-monitoring checkpoint systems that ensure correct temporal and spatial coordination of chromosome segregation by the microtubule spindle apparatus. Defects in these checkpoint systems can lead to genomic instability, an important aspect of tumorigenesis. Here we show that the von Hippel-Lindau (VHL) tumour suppressor protein, pVHL, which is inactivated in hereditary and sporadic forms of renal cell carcinoma, localizes to the mitotic spindle in mammalian cells and its functional inactivation provokes spindle misorientation, spindle checkpoint weakening and chromosomal instability. Spindle misorientation is linked to unstable astral microtubules and is supressed by the restoration of wild-type pVHL in pVHL-deficient cells, but not in naturally-occurring VHL disease mutants that are defective in microtubule stabilization. Impaired spindle checkpoint function and chromosomal instability are the result of reduced Mad2 (mitotic arrest deficient 2) levels actuated by pVHL-inactivation and are rescued by re-expression of either Mad2 or pVHL in VHL-defective cells. An association between VHL inactivation, reduced Mad2 levels and increased aneuploidy was also found in human renal cancer, implying that the newly identified functions of pVHL in promoting proper spindle orientation and chromosomal stability probably contribute to tumour suppression.

Sporadic clear cell renal cell carcinoma but not the papillary type is characterized by severely reduced frequency of primary cilia

Renal cysts and clear cell renal cell carcinoma are common clinical manifestations of people with germ-line mutations of the von Hippel–Lindau tumor suppressor gene, VHL. Recent cell biological evidence suggests that the VHL gene product, pVHL, functions to maintain the primary cilium, a microtubule-based antenna-like structure whose functional integrity is believed to have an important role in cell-cycle control. As VHL mutations are common in sporadic clear cell renal cell carcinoma, but not papillary renal cell carcinoma, we asked whether there is an association between VHL status and primary cilia in vivo. VHL status was assessed in 20 cases of clear cell renal cell carcinoma and 9 cases of papillary renal cell carcinoma by DNA sequencing and by immunohistochemical staining for the hypoxia-inducible factor-α target gene products CA9 and GLUT-1. Of 20, 18 clear cell renal cell carcinomas, but only 1 of 9 papillary renal cell carcinomas, displayed evidence of VHL inactivation. In clear cell renal cell carcinoma the frequency of ciliated tumor cells ranged from 0 to 22% (median value 7.8±6.0%), whereas cilia frequency was significantly higher (P<0.0001) in papillary renal cell carcinoma (range 12–83%, median value 43.3±21.3%). There was no correlation between Ki-67 staining and cilia frequency, suggesting that the observed differences between the tumor types in cilia frequency are not accounted for by differences in cellular proliferation rates and that primary cilia degeneration in sporadic clear cell renal cell carcinoma depends on VHL inactivation. We propose that the different ciliation status of clear cell and papillary renal cell carcinoma may contribute, at least in part, to the different biological behaviors of these tumor types.

Priming-Dependent Phosphorylation and Regulation of the Tumor Suppressor pVHL by Glycogen Synthase Kinase 3

ABSTRACT Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is linked to the development of tumors of the eyes, kidneys, and central nervous system. VHL encodes two gene products, pVHL30 and pVHL19, of which one, pVHL30, associates functionally with microtubules (MTs) to regulate their stability. Here we report that pVHL30 is a novel substrate of glycogen synthase kinase 3 (GSK3) in vitro and in vivo. Phosphorylation of pVHL on serine 68 (S68) by GSK3 requires a priming phosphorylation event at serine 72 (S72) mediated in vitro by casein kinase I. Functional analysis of pVHL species carrying nonphosphorylatable or phosphomimicking mutations at S68 and/or S72 reveals a central role for these phosphorylation events in the regulation of pVHLs MT stabilization (but not binding) activity. Taken together, our results identify pVHL as a novel priming-dependent substrate of GSK3 and suggest a dual-kinase mechanism in the control of pVHLs MT stabilization function. Since GSK3 is a component of multiple signaling pathways that are altered in human cancer, our results further imply that normal operation of the GSK3-pVHL axis may be a critical aspect of pVHLs tumor suppressor mechanism through the regulation of MT dynamics.

Quantitative image analysis identifies pVHL as a key regulator of microtubule dynamic instability

The product of the von Hippel-Lindau tumor suppressor gene stabilizes microtubules by inhibiting GTPase activity.

The VHL Tumor Suppressor: Riding Tandem with GSK3β in Primary Cilium Maintenance

Amongst other clinical manifestations, patients with the von Hippel-Lindau (VHL) cancer syndrome are predisposed to develop kidney cysts, which are considered to be precursor lesions of clear cell renal cell carcinoma (ccRCC). Recent evidence has highlighted an unexpected function of the VHL tumour suppressor protein (pVHL) in maintaining the structural integrity of the primary cilium, a microtubule-based cellular antenna important for suppression of uncontrolled proliferation of kidney epithelial cells and cyst formation. Intriguingly, this function of pVHL is directly linked to its capacity to regulate the microtubule cytoskeleton independent of its well-characterized role in the degradation of hypoxia inducible factor alpha (HIFα) subunits. However, loss of pVHL alone does not suffice for a cell to lose the primary cilium. Other pathways need to be additionally inactivated, including one involving glycogen synthase kinase 3 beta (GSK3β). These new findings draw attention to a primary cilium-maintenance network as new territory for pVHL tumour suppressive activity and have implications for understanding the development of kidney pathology in the setting of VHL disease.