Defining the role of 1433 sigma in cancer progression

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Defining the role of 14-3-3 sigma in cancer
progression

• Bruce J. Herron
• Wadsworth Center NYS Department of Health
• School of Public Health SUNY Albany

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Goals of lecture

• Determine how 14-3-3 sigma (sfn) contributes to
  cancer in vivo
• describe a novel application of genomics to
  determine relationships between IKKa and sfn
• propose a model for Sfns role in cancer
  progression based on observed outcomes

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Functional annotation of mammalian genomes
Waved 1 Tgf alpha

• Assignment of gene function based on phenotypic
  measures.
• Model systems that employ genetic screens often
  use similar phenotypes in distinct loci to
  associate them in a common functional category.

Waved 2 EGF receptor
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Terminal differentiation
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animal models of cancer

• Observe interaction between cells and genes
• genetics
• developmental biology
• Today's model Repeated epilation
• Sfn deficient

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Repeated Epilation (ER)

• 1month old (Er/)
• Repeated loss of hair, Longer nails
• 1 year old (Er/)
• Skin cancer

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Recessive phenotype perinatal lethal
Recessive Er/Er
WT
Er/Er
WT
Er/Er
WT
Er/Er
WT
Er/Er
PECAM
Herron etal Nature Genetics 37, 1210 - 1212
(2005)
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Skeletal defects
Normal
Er/Er
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A mutation in stratifin is responsible for the
repeated epilation (Er) phenotype in mice.
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14-3-3 proteins

• Seven member family in mammals
• Bind phosphorylated targets
• Mediate activity, localization, stability

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Sfn in cancer

• Repressed via methylation in tumors
• 50 - 100 of tumors analyzed
• Mediator of P53-induced DNA damage response
• Does Sfn only mediate Cell cycle arrest in cancer?

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Sfn has a specific function in epithelia
Stratum corneum Loricrin Filagrin Granular Layer
K1/K10 Spinous Layer K5/K14 Basal Layer

• Highly expressed in differentiated keratinocytes
• Deletion induces stem cell-like phenotype.

Lor
K10
K5
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One phenotype three genes
Er/Er
IKKalpha
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I-Kappa Kinase a null

• Classic function upstream kinase of NfKB
• Co kinase IKKbeta required for NfKB-specific
  functions
• IKK alpha null is very similar to sfn mutant
  phenotype
• What are the connections between these two
  phenotypes?

Abnormal Morphogenesis But Intact IKK Activation
in Mice Lacking the IKK1 Subunit of I B Kinase
Yinling Hu, 1 Véronique Baud, 1 Mireille
Delhase, 1 Peilin Zhang, 2 Thomas Deerinck, 3
Mark Ellisman, 3 Randall Johnson, 4 Michael Karin
Science, Vol 284, Issue 5412, 316-320 , 9 April
1999
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Development IKKa deficient mice
IKKa deficient embryonic stem cells were obtained
from the Genetrap consortium (www.genetrp.org).
Mice homozygous for the genetrap insertion were
indistinguishable from IKKa knock out mice.
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Sfn and IKKa have similar defects in skin
differntiation
Er/Er
Stratum corneum Loricrin Filagrin Granular layer
K1/K10 Spinous Layer K5/K14 Basal Layer
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No direct relationship between Sfn and IKKa
IKKa deficient
Er/Er
WT
anti-Sfn
anti-IKKa
anti-beta actin
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Genomic analysis of sfn and IKKa

• Embryonic day 18.5 full thickness skin
• Littermate controls (same genetic background)
• GCRMA normalization
• Bonferroni corrected greater than 3 fold

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Expression profiles
sfn
WT
IKKa

• More than 2000 expressed differences between
  mutants and controls (gt3 fold)
• IKKa and Sfn have many unique expression
  differences

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Sfn (Er/Er) is distinct from IKKalpha and IRF6
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Cdkn1a cyclin-dependent kinase inhibitor 1A
(p21)
IKKa deficient
Er/Er
/
anti-p21
anti-ßactin
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Gene ontology

• Hierarchical description of characterized gene
  function
• Genes with common function assigned to specific
  categories
• Can help to develop hypotheses about the basis
  for a phenotype
• Requires follow up experiments to confirm
  findings
• Cannot help wit uncharacterized genes

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Commonly changed GO in both IKKa deficient and
Er/Er

sfn
WT
IKKa
Genespring 10.0 The p-value for each GO term
reflects the enrichment in frequency of that GO
term in the input entity list relative to the All
Entities list.
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Gene Ontology Analysis
sfn
WT
IKKa
Genespring 10.0 The p-value for each GO term
reflects the enrichment in frequency of that GO
term in the input entity list relative to the All
Entities list.
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Expression of all cell cycle genes
sfn
WT
IKKa
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Skin proliferation
nonproliferative
Proliferative
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Er/Er and IKKa deficient skin have defects in
differentiation-induced apoptosis
Er/Er
/
IKKa deficient
Tunnel
200X
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Cell cycle in primary keratinocytes
S-phase
BrdU incorporation
G1
G2
2N
4N
DNA Content/ cell analyzed
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Er/Er keratinocytes undergo cell cycle arrest in
high calcium media
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Sfn keratinocytes have abnormal DNA content
IKK alpha
Er/Er
Wild Type
Sfn
Low Calcium (proliferating)
2N 4N 8N
2N 4N 8N
High Calcium (Differentiated)
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Do Sfn keratinocytes have defects in cytokinesis?
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Are translational defects associated with Er/Er
skin abnormalities?
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Model for dual control of skin differentiation
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Genes associated with epidermal differentiation
are expressed in Er/Er skin
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mRNA versus protein of cornified envelope protein
loricrin
IKKa deficient
Er/Er
sfn
WT
Lor
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How do we determine if an mRNA is translated?

• Polysomes
• a group of ribosomes joined by a molecule of
  messenger RNA
• mRNAs undergoing translation are treated with
  cycloheximide and isolated via sucrose density
  gradient

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Post transcriptional control of protein expression

• mammalian target of rapamycin (mTOR)
• senses and responds to nutrient availability,
  energy sufficiency, stress, hormones and mitogens
  to modulate protein synthesis
• Active mTOR facilitates growth and proliferation
  though the induction of Cap-dependant
  translation.
• 14-3-3 proteins are known to interact with
  several mediators of translational control

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Xiaoju Max Ma and John Blenis Molecular
mechanisms of mTOR mediated translational
control NATURE REVIEWS Molecular cell Biology
May 2009
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(No Transcript)
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Inhibition of mTOR restores Loricrin expression
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Summary

• Sfn deficiency in Er/Er mice lead to severe
  defects in differentiation
• While phenotypically similar to IKKalpha skin,
  Er/Er skin has distinct expression patterns that
  reflect biological differences
• While sfn is known to mediate G2/M progression
  sfn deficiency in Er/Er skin does not
  substantially impact proliferation
• The role of sfn deficiency in cancer progression
  may be to prevent the switch from cap-dependant
  to cap independent translation.

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Unanswered questions/ Ongoing studies

• How do some proteins escape translational
  repression in sfn-deficient skin?
• What is the target of sfn in translational
  repression?
• Is there a relationship between translational
  repression and sfn status in human tumors?
• Should rapamycin be used as a therapeutic option
  in tumors with sfn repression?

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Acknowledgements

• Wadsworth
• Fang liu
• Jason Smith
• Barbara Beyer
• Hongliu Sun
• Zhen Zang
• Michigan State
• Brian Schutte

• NYNSCI
• Gretchen Kusek
• SUNY Albany
• Tom Begley
• Funding
• NIH/NIAMS
• New York State Office of Technology and Academic
  Research