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New insights into an enigmatic


tumour suppressor
Peter J. Ratcliffe

Dynamic regulation of the microtubule apparatus is central to cell division, cell migration, polarity and
transport. New data demonstrating functional association of the von Hippel-Lindau (VHL) tumour
suppressor with microtubules provide a new lead in unravelling VHL tumour suppressor mechanisms.

on Hippel-Lindau disease is a heredi-

V tary cancer syndrome that predispos-


es people to tumours of the kidney
(renal cell carcinoma (RCC)), retina and
Type 2A VHL disease mutants

pVHL
Type 2B VHL disease mutants

pVHL
central nervous system (haemangioblas-
toma (HB)), the adrenal gland (phaeochro-
mocytoma) and various cystic lesions
mainly affecting the kidney and pancreas
(for review, see ref. 1). Despite identifica- HIF-α HIF-α
tion of the VHL gene on chromosome 3p25
almost ten years ago, the tumour suppres- MT stabilization MT stabilization
sor mechanism remains poorly understood. HIF target function disrupted HIF target function intact
On page 64 of this issue, Hergovich et al. genes 'up' genes 'up'
now provide data indicating a new facet to
VHL function2. They demonstrate associa-
tion of the VHL gene product with the
microtubule apparatus and suggest that Low risk of RCC High risk of RCC
mutations interfering with a microtubule-
stabilizing function of VHL may contribute
to the complex genotype–phenotype rela- Figure 1 Model for different VHL functions and the risk of RCC disease sub-type.
tionship that is observed in VHL disease.
VHL disease is one of a number of dom-
inantly inherited cancer syndromes (other also unusual4. Re-expression of the wild- tumour suppressor mechanisms.
examples include familial retinoblastoma, type VHL in VHL-defective RCC lines had To what extent can these findings be
Li-Fraumeni syndrome and familial adeno- no effect on monolayer growth but reduced explained by current knowledge of the
matous polyposis affecting the Rb, TP53, growth of such cells as tumours in nude molecular function of pVHL? To date, VHL
and APC genes, respectively) in which mice. This has indicated that the tumour is best understood as a ubiquitin ligase in
molecular analysis has confirmed the pre- suppressor action is in some way dependent the oxygen-dependent proteolytic degrada-
dictions of Knudson’s classic ‘two-hit’ on conditions manifested during solid tion of the hypoxia inducible factor (HIF)
hypothesis. In such syndromes, affected tumour growth, but not during standard transcriptional complex8. Although a criti-
families manifest germline transmission of a monolayer cell culture, fuelling interest in cal physiological role for VHL in hypoxic
single predisposing mutant allele, with understanding abnormalities of cell–matrix gene regulation is established, the extent to
somatic inactivation of the second allele and cell–cell interactions in these cells5–7. which dysregulation of the HIF pathway
being required for cancer development. In The complex nature of VHL function is accounts for cancer predisposition in VHL
general, functional analyses of the mutated further suggested by the fact that clinical disease remains uncertain. Support for a
tumour suppressor genes in such diseases observations of VHL disease demonstrate a tumour-promoting effect has been
have also conformed well to the predictions complex genotype–phenotype relation- obtained (at least for one HIF isoform,
of classic cancer genetics. The relevant gene ship. Families have been classified as type 1 HIF-2α) from rescue of the tumour sup-
products have usually been shown to have or type 2, according to whether the risk of pressor effects of VHL by a mutant HIF-2α
clear roles in the control of cell proliferation phaeochromocytoma is low or high1 (Table that escapes VHL-mediated proteolysis9.
or in genetic repair processes, and thus have 1). Definition of the mutational spectrum Nevertheless, direct mutational activation
been classified as gatekeepers or caretakers, has indicated a clear bias against inactivat- of HIF has not, so far, been described in rel-
respectively. Unusually, however, the VHL ing mutations in type-2 disease, suggesting evant tumour types. This has suggested that
tumour suppressor has not yet been clearly either that the development of phaeochro- oncogenic selective pressures might in fact
accommodated within this model. The large mocytoma is incompatible with complete be operating on different, as yet unknown,
number of benign multicellular lesions that loss of VHL function, or that some gain of VHL-associated pathways.
are found in affected organs has suggested a function is acquired by VHL mutations In attempts to define such pathways, sev-
gatekeeper mechanism. Nevertheless, that promote this type of tumour. Further eral studies have compared gene expression
indices of proliferation in such lesions are complexity is added by striking differences profiles and cellular phenotypes in VHL-
relatively low3. Moreover, the findings from in the risk of RCC and HB among families defective RCC cells and transfectants re-
direct assays of later aspects of VHL tumour bearing type-2 mutations (Table 1). Taken expressing wild-type VHL. Such studies have
suppressor function, in which VHL is re- together, these findings have suggested the implicated VHL in a variety of processes of
introduced into fully transformed cells, are operation of distinct, possibly tissue-specific, potential relevance to its tumour suppressor

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activity. These include cell cycle regulation function is independent of E3 complex for- However, irrespective of whether this precise
by exogenous stimuli, extracellular matrix mation, suggesting that microtubule binding explanation holds up, the new finding that
formation, cell–cell interactions, cell inva- and stabilization may be an independent VHL associates with the microtubule appa-
sion, branching morphogenesis and epithe- function of VHL. ratus is convincing and provides an impor-
lial organization5–7,10,11. A key question is To address the potential relationship to tant lead in understanding VHL function.
whether these properties are connected with tumour suppressor function, they asked how Interestingly, another tumour suppressor
each other or with the hypoxia response, or VHL mutations associated with different (the adenomatous polyposis coli, APC gene
whether they represent distinct actions of patterns of tumour development affect product) is known to interact with the
VHL that might reveal distinct tumour sup- microtubule stability. This approach has pre- microtubule apparatus, and existing data
pressor pathways. viously been used to analyse links between on the APC–microtubule interaction
How does the new work by Hergovich et dysregulation of HIF and VHL disease sub- could be of interest in directing new stud-
al. fit with these findings? Using a combina- types and has supported a role of HIF acti- ies of VHL. Similarly to VHL, APC has a
tion of approaches, they revealed that cyto- vation in HB and RCC, but not phaeochro- well-defined proteolytic function, regulat-
plasmic VHL is largely associated with the mocytoma (see Table 1)12,13. In the current ing the Wnt signalling pathway through
microtubule apparatus. A complex twist to study, Hergovich et al. found that although the ubiquitin-mediated degradation of
this tale is that this association was specific many missense mutations retained the abili- β-catenin. However, accumulating evi-
to one VHL isoform only. Human pVHL ty to stabilize microtubules, at least under dence has indicated additional or connect-
exists as two isoforms: a protein with a rela- assay conditions explored, this property was ed roles for APC in microtubule functions.
tive molecular mass (Mr) of approximately abrogated by two specific mutations, Y98H APC promotes microtubule polymeriza-
30,000 (30K; pVHL30) that corresponds to and Y112H. These mutations are associated tion in vitro and in vivo and preferentially
the predicted 213-amino-acid product of with type-2A disease (a high risk of localizes to the plus (growing) ends via
the entire open reading frame and a second phaeochromocytoma and a low risk of RCC, associations with another micotubule-
19K isoform (pVHL19) produced by transla- see Fig. 1), a genotype–phenotype pattern associated protein EB1 (ref. 14). Although
tional initiation at an internal methionine at that is quite distinct from the effects of VHL the role of defective microtubule functions
codon 54. The pVHL19 isoform is competent mutations on HIF. One possibility is that in APC tumour suppressor behaviour is
for ubiquitin ligase activity1, and, as all defective microtubule stabilization might unclear, localization studies have stimulat-
tumour-associated mutations affect pVHL19 contribute to the development of ed two lines of enquiry. In mitotic cells,
coding sequences, it has been assumed that phaeochromocytoma. However, if this is the APC–EB1 complexes localize to the spin-
this function is critical for tumour suppres- case, it is difficult to understand why alterna- dle–kinetochore connection and it has
sor function. tive substitutions at Tyr 98 and Tyr 112 that been proposed that defective attachment
Using two-colour confocal immunos- are associated with type 2B disease (a high results in a chromosomal instability pheno-
taining, Hergovich et al. show differential risk of phaeochromocytoma and RCC, see type that is observed in APC-defective cells15.
localization of pVHL19 and pVHL30 iso- Fig. 1) were found to behave normally in In interphase cells, APC has been shown to
forms, demonstrating that although pVHL19 these assays and why mutations that affect cluster at the plus ends of microtubules near
is predominantly localized to the nucleus, pVHL19 seem to be necessary for tumour- the plasma membrane. The association of
the longer pVHL30 isoform is largely local- promoting effects. This suggests a different these clusters with the formation of cellular
ized to the microtubule apparatus. In addi- explanation — that the mutational effect extensions and alterations in the response to
tion, the localization of pVHL30 is markedly somehow mitigates against the RCC predis- ‘wound healing’ of confluent cultures have
perturbed by drugs that alter microtubule position. How can this hypothesis be suggested an involvement in the regulation
polymerization. The authors demonstrate a squared with the high risk of RCC associat- of cell motility16. Given the existing focus on
physicial association of VHL with micro- ed with completely inactivating VHL muta- abnormal cell–matrix interactions and
tubule proteins by cosedimentation with tions? The authors noted that although the epithelial organization in VHL-defective
taxol-stabilized microtubule assemblies and Y98H and Y112H mutants are dysfunctional cells, the new data demonstrating a
by co-immunoprecipitation of α-tubulin in microtubule stability assays, they continue VHL–microtubule association provides an
with VHL. Moreover, using in vivo assays of to bind the microtubule apparatus, suggest- interesting new lead in the pursuit of this
microtubule stability, they show that VHL ing that the low risk of RCC may represent a enigmatic tumour suppressor function.
protects against nocodazole-induced micro- gain-of-function effect of these mutations. Peter J. Ratcliffe is at the The Henry Wellcome
tubule dissassembly2. Interestingly, from More work will be needed to prove such Building of Genomic Medicine, Roosevelt Drive,
mutational analysis, they found that this an effect and to define the mechanism. Oxford, OX3 7BN, UK
pjr@well.ox.ac.uk
1. Kaelin, W. G. Nature Rev. Cancer 2, 673–682 (2002).
2. Hergovich, A. et al. Nature Cell Biol. 4, 64–70 (2002).
3. Mandriota, S. J. et al. Cancer Cell (in the press).
Table 1 Genotype–phenotype associations in VHL disease 4. Iliopoulos, O., Kibel, A., Gray, S. & Kaelin, W. G. Jr. Nature
Med. 1, 822–826 (1995).
Type 1 Type 2 5. Koochekpour, S. et al. Mol. Cell. Biol. 19, 5902–5912 (1999).
A B C 6. Ohh, M. et al. Mol. Cell 1, 959–968 (1998).
7. Davidowitz, E. J., Schoenfeld, A. R. & Burk, R. D. Mol. Cell.
Tumours Biol. 21, 865–874 (2001).
Phaeochromocytoma – + + + 8. Maxwell, P. H. et al. Nature 399, 271–275 (1999).
9. Kondo, K. et al. Cancer Cell 1, 237–246 (2002).
RCC + – + – 10. Bindra, R. S. et al. Cancer Res. 62, 3014–3019 (2002).
HAB + + + – 11. Esteban-Barragan, M. A. et al. Cancer Res. 62, 2929–2936
(2002).
12. Clifford, S. C. et al. Hum. Mol. Genet. 10, 1029–1038 (2001).
Funtional assays 13. Hoffman, M. A. et al. Hum. Mol. Genet. 10, 1019–1027 (2001).
14. Nakamura, M., Zhou, X. Z. & Lu, K. P. Curr. Biol. 11,
HIF proteolysis – – – + 1062–1067 (2001).
MT stabilization ? – + + 15. Pellman, D. Science 291, 2555–2556 (2001).
16. Mimori-Kiyosue, Y., Shiina, N. & Tsukita, S. J. Cell Biol. 148,
505–517 (2000).

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