Lester and Sue Smith Breast Center Journal Club

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Lester and Sue Smith Breast CenterJournal Club

• Bonita Chan
• Adrian Lee Laboratory
• Feb 3, 2009

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Loss of Cell Polarity during the Course of
Breast Carcinoma
Lee et al., Breast Cancer Res 2006
Hebner et al., Annu Rev Pathol Mech Dis 2008
Dimri et al., Breast Cancer Res 2005
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Scribble a polarity protein
Macara, Nat Rev Mol Cell Biol, 2004

• A member of the LAP proteins
• Leucine-rich-regions (LRR)
• PSD-95/Dlg/ZO-1 (PDZ) domains

Macara, Nat Rev Mol Cell Biol, 2004
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Scribble and other polarity proteins in human
cancers

• Human Scribble and DLG1 are targeted for
  ubiquitin-mediated proteolysis by the E6
  onocoprotein from high-risk strains of human
  papillomavirus (Gardiol et al., 1999 Humbert et
  al., 2003 Nakagawa and Huibregtse, 2000)
• Genetic alteration in dlg5 is correlated with
  inflammatory bowel disease, which predisposes the
  patient to gastric cancer (Stoll et al., 2004)
• Loss of Scribble expression is frequently
  observed in colon and lobular breast cancer
  (Gardiol et al., 2006 Navarro et al., 2005)

Rosen and Vargo-Gogola, Nat Cancer Rev 2007
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Scribble loss blocks apoptosis and alters 3D
organization of epithelial cells
Comparable acini observed.
Disruption of Golgi orientation (apical-basal
polarity).
Lumen filling (gt55 in Scrib. RNAi vs. lt6 in
control)
Stable depletion of Scribble by RNAi - No change
in cell morphology (?-catenin)
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Reduced luminal cell death by Scrib. RNAi.
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Loss of Scribble promotes abnormal ductal
morphogenesis in transgenic mammary glands
COMMA-1Dßgeo (CD)-GFP cells repopulate in
epithelial-free mammary fat pad and form
transgenic mammary ducts (41 weeks after
transplantation).
Dense and enlarged end buds found in the
transplanted mammary fat pad (as soon as 8 week
of transplantation).
Suppression of Scribble by RNAi in CD-GFP cells.
Normal glandular structures in control vs.
multilayered epithelia with no apparent ductal
space in Scrib. RNAi transplanted mammary fat pad.
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Scribble loss blocks apoptosis in the context of
E7
Large, filled luminal acini found in Scrib. RNAi
cells.
Inhibited apoptosis.
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Scribble loss blocks apoptosis in the context of
Myc
Expression of Myc-ER in MCF-10A cells
gt45
lt15
Increased cell proliferation (Ki67) and cell
death (Act-Caspase-3) by activation of myc in
Myc-ER MCF-10A acini.
Loss of Scribble blocks c-Myc-induced apoptosis
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Scribble loss blocks apoptosis in the context of
Myc
Loss of Scribble suppressed c-Myc-induced Bim
expression
Rescue of Scribble function in Scrib. RNAi cells
restores sensitivity to Myc-induced upregulation
of Bim
Rescue of Scribble function in Scrib. RNAi cells
restores sensitivity to Myc-induced apoptosis.
(Scrib. RNAi expression was monitored by GFP
expression)
Disintergrating acini with abnormal morphology
(gt60) found in Myc-ER MCF-10A cells with long
term activation of myc.
Loss of Scribble with long term activation of
c-Myc resulted in formation of large disorganized
multiacinar structures.
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Myc induces activation of Rac by promoting a
Scribble-ßPIX/GIT1 interaction
Loss of Scribble suppressed Rac activity
Frank and Hansen, Cell Dev Biol 2008
Loss of Scribble suppressed c-Myc-induced
activation of Rac. Scribble is not required for
EGF-induced activation of Rac.
Activation of c-Myc in MCF-10A cells increases
formation of Scribble-ßPIX/GIT1 complex
Activation of c-Myc in MCF-10A cells induces ßPIX
and GIT1 mRNA levels
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Myc induces apoptosis by activating Rac-JNK-BIM
pathway
Inhibition of Rac or JNK blocked Myc-induced
apoptosis in 3D acini
Inhibition of Rac blocked Myc-induced BIM level
Activation of c-Myc induced increase in p-Jun (no
increase in cells with loss of Scribble)
Downregulation of ßPIX blocked c-Myc induced
apoptosis
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Loss of Scribble cooperates with Myc to induce
mammary tumors
Loss of Scribble inhibited c-Myc-induced
apoptosis, but not enhancing proliferation in
mammary tumors.
Loss of Scribble induced larger (10-fold) tumors.
Loss of Scribble significantly reduced the levels
of Bim and p-Jun in mammary tumors.
Loss of Scribble induced glandular, low grade,
and well-differentiated tumors.
Loss of Scribble is selected for during MMTV-Myc
mammary tumorigenesis.
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Mislocalization of Scribble phenocopies loss of
Scribble expression
Downregulation of Scribble mRNA level (gt 2-fold)
is found in 17 out of 32 breast tumors.
Mislocalization of Scribble in human breast
cancer cell line MCF-7 (cytoplasmic rather than
restricted to cell-cell junctions).
Mislocalization of Scribble in 10 out of 20 DCIS
samples mosaic for normal and mislocalized
Scribble (regions with normal Scribble
localization).
Mislocalization of Scribble and disrupted Golgi
orientation in MCF-10A cells with P305L mutant
(disrupted membrane binding of the LRR domain)
Mislocalization of Scribble suppressed the
myc-induced apoptosis.
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Summary

• Loss of Scribble expression
• Phenotype
• in normal epithelial cells
• Blocks apoptosis
• Disrupts Golgi orientation
• Induces abnormal morphogenesis in transgenic
  mammary glands
• in oncogene-expressing epithelial cells
• Blocks oncogene-induced apoptosis
• Mechanism
• Disrupts Myc pathway
• Suppresses Myc-induced Bim level (apoptosis of
  central cells to form lumen)
• Reduces Rac activity (Myc ? Scribble-ßPIX/GIT1 ?
  Rac)
• Blocks Rac-JNK-Bim ? Apoptosis
• Induces mammary tumors
• Mislocalization of Scribble phenocopies loss of
  Scribble expression

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Working Model
Myc and Scribble
HPV E7