Post on 08-Jan-2016
description
Pavel Yarmolenko
Biology 169, Spring 2005
The von Hippel-Lindau (VHL) Tumor Suppressor Gene Inactivation and Its
Involvement in Tumorgenesis
Overview• 1) von Hippel-Lindau disease – biallelic mutation/inactivation of VHL gene
• 2) Cell response to hypoxia – What VHL does.
• 3) pVHL role – the protein VHL gene codes for.– Its function– How it was studied– Knockouts– Possible treatment options
• 4) New Research, Future Directions
von Hippel-Lindau Disease• Hereditary cancer syndrome affects 1 in 35,000 individuals.
• Transmitted in an autosomal dominant manner.
• >150 different mutations in the VHL gene cause VHL disease.– Many of them are categorized by phenotype in humans.
• Sporadic tumors – both copies of VHL mutated after conception.
• Lesions associated with VHL disease include, among others:– CNS and retinal hemangioblastomas– clear-cell renal carcinomas– Pheochromocytomas
VHL Disease
Bottom Line
Highly vascular lesions that overproduce angiogenic, hypoxia-inducible markers (such as vascular endothelial growth factor (VEGF) and erythropoeitin (Epo))
Normal Cellular Response to Hypoxia
AngiogenesisECM dissolution
Migration
AngiogenesisDedifferentiation
ProliferationMigration
Modified from WY Kim and WG Kaelin. J Clin Oncol 22:4991-5004
What is HIF?
HIF Examined-It is a hypoxia-inducible transcription factor-Heterodimer formed by HIFα and HIFβ-HIFβ is a stable protein-HIFα is degraded in presence of O2
HIFα activity controlled by OXYGEN-DEPENDENT hydroxylation at either the ODDD or CTAD domain.
Bind to DNA and Activate Transcription
Bind HIF-1β
What controls HIF?
Control of HIF
Modified from WY Kim and WG Kaelin. J Clin Oncol 22:4991-5004
Ubiquitin Mediated Proteolysis
Courtesy of Dr. Robert Duronio
How it was figured out
What else does VHL do?
1988Mapped
by linkage analysis
1993Isolated
by positional cloning
1990
1. VHL = inactive in disease.2. Hypoxia-inducible mRNAs
overproduced.
1993-99 CBP/p300 coactivator recruitment studied
1993-99 VHL interaction studied
2000
2000
What controls HIFα?
HIF prolyl hydroxylase gene identified in
1999VHL complex
Identified(E3 Ubiquitin Ligase)
1999 CBP/p300and its action
identified
2000How does the ODDD make
HIFα unstable in high [O2]?
2002 FIH-1 and its action
identified
2001 EGLN hydroxylate
(Pro)n in ODDD
Altered Cell Cycle• pVHL -/- cells are unable to exit the cell cycle under certain experimental conditions,
such as growth in low serum. – Ability to exit the cell cycle is restored by reintroduction of wild-type pVHL.
• pVHL is a negative regulator of cyclin D1, which is a mitogen.
• pVHL also negatively regulates TGFα (HIF target gene). Renal tubular epithelial cells are very sensitive to the mitogenic effects of TGFα.
ECM Connection
• pVHL -/- cells do not deposit a proper extracellular fibronectin matrix
– pVHL +/+ cells restore the function.
– Epithelial cells exhibit increased proliferation and decreased differention when grown on specific matrices.
• Cells lacking pVHL are more invasive and secrete higher levels of matrix metalloproteases.– VEGF upregulates matrix metalloprotease synthesis, so this process is HIF-dependent.
Poor ECM More Tumor-like behavior of normal epithelial cells
Cytoskeleton Connection- 2003 Turcotte et al., Am J Physiol Renal Physiol. 2004 Feb;286(2):F338-48
2.5 x pVHL expression
Rho proteins (GTPases) that regulate the organization of the actin cytoskeletonDisruption of actin and microtubule
networks by chemicals or hypoxia
pVHL influences microtubule dynamics at the cell periphery independently of its effects on HIFα- done in cells that don’t accumulate HIFα- the cell line is tumorigenic in mouse xenografts – pVHL inactivation causes tumors without HIFα accumulation.
-2004 MP Lolkema et al., Experimental Cell Research 301 (2004) 139– 146
-2003 A Hergovich et al., Nat Cell Biol. 2003 Jan;5(1):64-70.
pVHL stabilizes microtubules through direct interaction.Does not require E3 Ubiquitin Ligase function
Mouse Knockouts• VHL-/- mice die during early embryogenesis.
• VHL+/- mice - normal life span, but may develop vascular tumors in the liver that overexpress HIF target genes.
– Conflicting reports on the above (tumors may or may not develop).
• Same lesions can be induced by inactivation of VHL in the liver.
Treatment Options
WY Kim and WG Kaelin. J Clin Oncol 22:4991-5004
Inhibition of HIF2α is sufficient to suppress VHL-/- tumor growth in vivo.-Kondo K et al., PLoS Biol 1:E83, 2003-Zimmer M et al., Mol Cancer Res 2:89-95, 2004
Unanswered Questions• Why is VHL upregulated in tumors?
• How does VHL affect secreted fibronectin?
• Why does VHL stabilize microtubules at the cell periphery while it’s present throughout the cytoplasm?
– Transduction pathway?
• What other non-HIF related functions does VHL have?
That’s IT!!!