Case Presentation

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Case Presentation

• 49 year old white female who presents for
  evaluation of liver tumors
• She has a history of retinal angiomas that
  developed when she was 13 years old. These have
  been treated with multiple laser surgeries and
  she is now blind on the left.
• She also has a history of an ACTH-producing tumor
  in the lung that was resected in 1996.

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Case continued

• ACTH producing neuroendocrine tumors were also
  found in the liver. These were treated with
  resection in 1998 and then radiofrequency
  ablation in 2001 for recurrent symptomatic
  lesions.

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Case continued

• Also history of renal cell carcinomas. These
  have been resected in 1996 then in 2001.
• Other history includes a history of BAD, and
  surgeries after trauma.
• Medications include synthroid, psychiatric drugs,
  darvocet, celebrex.

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Case continued

• Family history is significant for a father who
  died of heart disease at 81 and a mother who died
  of dementia at 73. She has 8 healthy sibs. She
  has one child 25 y/o with spina bifida, one with
  IDDM at 14 y/o, and a 20 y/o with learning
  disability.
• She lives with her husband and two children. She
  has 30 pack year of tobacco and quit 3 years ago.
• She drinks occasional alcohol and works in the
  home now.

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Physical exam

• Obese white female with striae over her abdomen
  and axillae.
• P92 BP137/83 Wt 183 lb Afebrile
• Surgical scars on right thorax and on the abdomen
  across the midline to the right.
• Pupils with surgical changes bilaterally, left
  eye deviated laterally.
• Otherwise, unremarkable.

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Radiographic Data

• CT of chest, abdomen, and pelvis remarkable for
  several arterial enhancing liver lesions. Also
  there is a pancreatic cyst and a mass consistent
  with a microcystic cystadenoma.
• Kidneys have multiple bilateral renal cysts.

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Von Hippel-Lindau Syndrome

• Autosomal dominant tumor with multiple benign and
  malignant tumors in multiple organ systems.
• Hallmarks include retinal angiomas, cerebellar
  and spinal hemangioblastomas, and renal cell
  carcinomas.
• Also see cystic lesions in the kidneys, pancreas
  and epididymitis.
• Adrenal pheochromocytomas occur in 7-20 of those
  affected. The presence of pheos defines the
  disease as Type 2.

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VHL

• Prevalence is around 1/40,000 in Western European
  countries.
• Affects sexes equally and is seen in all ethnic
  groups.
• Penetrance is estimated at 80-90 by 65 years
  old.
• Diagnosed at infancy thru seventh decade.

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Diagnostic Criteria

• Single retinal or cerebellar hemangioblastom,RCC,
  or pheo is sufficient in someone with a known
  family history.
• In an isolated case, diagnosis is made with 2 or
  more hemangioblastomas or a single
  hemangioblastoma and a characteristic visceral
  tumor.
• Type I--No pheo
• Type 2A pheo without RCC and 2B with both.

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Molecular Basis

• Positional cloning in families with overlapping
  germline deletions led to the identification of
  the VHL gene on 3p25-26.
• VHL is a tumor suppressor gene that follows
  Knudsons 2-hit hypothesis. In VHL families, the
  wild-type allele was lost in VHL tumors.

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VHL gene product

• 213 amino acids with a molecular mass of the
  protein of approx. 20-30kDa
• mRNA expressed in all adult tissues.
• Mutations are heterogeneous. 15-20 have large
  deletions. 27 with missense and 27 with
  framshift or nonsense mutations.
• Mutations identified in nearly all VHL families.
• In Type 2- 96 have missense whereas deletion and
  premature termination is associated with I.

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VHL protein

• Binds Elongin B and C. Elongin B and C are
  components of a transcriptional elongation
  complex called Elongin/SIII

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pVHL and Ubiquitination

• pVHL forms stable complexes with the elongins and
  Cul2 and Rbx1. Cul2 and elongin C showed
  homology to yeast E3 ubiquitin ligases that were
  involved in substrate recognition for
  ubiquitination.
• Is pVHL involved in ubiquitination?

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Ubiquitination Review(Simplified)

• Ubiquitin uses ATP to form a thiol ester
  intermediate with E1-ubiquitin activating enzyme.
• Activated ubiquitin is then transferred to E2
  from E1.
• In the presence of E3, E2 transfers ubiquitin to
  the specific substrate.
• Polyubiquitinated proteins are degraded by the
  26S proteasome.

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IP VHL complex. Inc. with E1, 32P UB, indicated
E2 and ATP regen system.
VBC-Cul 2 is necessary for Ub
See more Ub w/ full-length pVHL
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Infect insect cells with E3 components. Use Ub
system. Mutants found in VHL pts failed to Ub
p220 and p100.
See components expressed
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Conclusion

• pVHL in the E3 complex is necessary for
  ubiquitination of p220 and p100.
• Mutations found in patients abrogates this affect.

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VHL and Hypoxia

• VHL tumors are usually very vascular and
  overproduce VEGF. They can also overproduce epo.
  Since VEGF and Epo are induced by hypoxia, is
  VHL involved in hypoxia regulation?

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Transfection of VHL into cell line lacking VHL
restores hypoxia-inducibility.
Controls not influenced by hypoxia or VHL.
mRNA analysis
pVHL is important for hypoxia-inducibility of
some RNAs
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Hypoxia-Inducible Factor

• Heterodimeric transcription factor consisting of
  HIF-1 ? and HIF-1 ?.
• Induces transcription of glucose transporters,
  glycolytic enzymes, and VEGF as well as cell-type
  specific proteins such as erythropoietin,
  inducible nitric oxide synthase, and IGF-2.
• HIF1- ? is required for normal vascularization,
  development, and survival of mouse embryos as
  well as for physiologic responses to chronic
  hypoxia in adult mice.

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HIF-1 is more stable in 1 O2. Degradation
mechanism saturated at high levels of expression
of HIF. Co-expression of VHL promotes the
degradation of HIF in 1O2
The proteasome inhibitor, MG-132 inhibits
degradation of HIF in the presence of VHL.
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Complex from infected insect cells was purified.
pVHL containing E3 mediates Ub of HIF-1 but not
another protein.
Ub of HIF requires hUbc5a.as E2
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Mutant pVHL do not form complex with elongins.
Mutant pVHL does not Ub HIF. pVHL is required.
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Hypoxia-Inducible Factor cont.

• Under normoxic conditions HIF-1? is
  post-translationally hydroxylated at proline
  residues.

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A hydroxylated proline peptide inhibits VHL HIF
interaction.
Wild-type but not mutant pVHL is captured by
OH-proline peptide.
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Our Patient

• Atypical in that she has an ACTH expressing
  tumor. Pathologically this resembles a
  paraganglioma.
• She is followed by CT scan to assess growth of
  liver nodules and development of renal cell
  carcinoma.
• She has had stable to decreased disease over the
  last year and a half.