Transcript Sox4 Links Tumor Suppression to Accelerated Aging in Mice

Sox4 Links Tumor Suppression to Accelerated Aging in
Mice by Modulating Stem Cell Activation
M. Foronado, P. Martinez, S. Schoeftner, G. Gomez-Lopez, R.
Schneider, J.M. Flores, D.G. Pisano, and M.A. Blasco
Cell Reports 8: 1-14 (2014)
Intro: What was known before the work described in the paper?
-adult organs maintained through balance of proliferation,
differentiation, and self-renewal of stem cells
-alterations in the balance between these in cancer and aging
Epidermis:
Interfollicular Epidermis (IFE)
Sebaceous Glands (SG)
Hair Follicles (HF)
Dividing Cells:
Hair Follicle Stem Cells (HFSC)
in Hair Bulge and HG
Dermis secretes signaling
molecules that regulate
HFSC proliferation,
differentiation, and self-renewal
through Wnt/β catenin pathway
and Sox4 transcription factor
Fig. 7.1
Sox4 transcription factor
-belongs to family of SRY box-containing transcription factors
that bind and induce bending in specific DNA sequences
-expressed mainly during embryogenesis in mesenchymal cells
-in adults, expression & function
is restricted to progenitor cell
populations in neuronal, cardiac,
immune and blood cell lineages
-overexpressed in all major
cancer types, correlates with
metastatic potential
-Sox4 null mutant mice die
in utero at day 14.5
New Question? What phenotype results from Sox4-knockout in a
specific tissue only (conditional Knock Out or cKO)?
Materials & Methods: Were any new materials or methods
developed specifically for this study? Why were they needed?
Mouse conditional knock-out technology through Cre/loxP System
Sox4lox/lox
Skin HFSC or HG cell
Sox4
Sox4cKO
in skin
progenitor
cell
Cre is a recombination enzyme that acts specifically at loxP sites.
Mouse cell-type specific promoters used to drive expression in a
specific tissue where gene deletion is desired. Other tissues remain
non-mutant, avoiding whole animal lethality.
How were the Sox4lox/lox and Cre Transgenic Mice Made?
Add DNA w/
selectable
marker
(inserts into
genome in
1/104 ES cells)
Blastocysts w/
transfected ES
cells develop into
adults, some
carrying transfected
DNA in GERMLINE
Fig. 18.49
Remove ES cells
from Blastocyst
and culture in vitro
Inject transfected
ES cells into Blastocyst
and implant in mouse
Results: What question is being addressed in each
figure, what experiment was used to answer that
question, and how were the results interpreted?
Fig. 1 Sox4lox/lox Mice Display
Cancer Resistance & Accelerated
Aging
Question: Phenotype in Sox4lox/lox
mice (knock-down intermediate)?
A)
B)
C)
D)
E)
F)
G)
H)
I)
J)
YES
Strategy for making Sox4lox/lox
PCR confirmation that it was made
Sox4lox/lox smaller in size
Sox4lox/lox smaller in weight
Sox4lox/lox has lower survival in wks
and Humane End Point
Sox4lox/lox has lower Bone Density
Sox4lox/lox has higher Hernia Rate
Sox4lox/lox has lower mean telomere
length in blood cells
Sox4lox/lox has lower spontaneous
cancer rate
Reduced Sox4 expression in all
tissues of Sox4lox/lox (lox sites flanking
gene must interfere with its transcription)
Fig. 2 Normal Skin Stratification in Sox4cKO, but Bulge Cells Have Reduced Replicative History
A)
B)
C)
D)
E)
Normal hair coat appearance
Sox4 expression absent in SoxcKO mice
Expression of skin-specific differentiation genes normal
Telomeres longer in HFSCs in Bulge of Sox4cKO mice (reduced replicative history)
5% HFSC telomeres < 50 a.u.f.; 95% > 800 a.u.f.
Fig. 3 Sox4 is Induced after Hair Plucking and Required for Normal Hair Regeneration
A) Induced genes following hair plucking include Sox 9
B) and Sox 4
C) Hair regeneration reduced in Sox4cKO relative to Sox4wt
Fig. 4 Sox4 is Required for
Normal HFSC Activation during
Hair Regeneration and
Wound Healing
A-H) Decrease in proliferating cells
as evidenced by decreased Ki67
(proliferation marker-rRNA txn) &
increased γH2AX, p53
(cell cycle arrest markers) in Sox4cKO
I) Reduced number of large,
differentiated colonies in
cultured keratinocytes from
Sox4lox/lox or Sox4cKO mice
J) Reduced wound healing in
SoxcKO mice
Fig. 5 Sox 4 is Required for
Induction of Proliferative and
Differentiation Pathways during
Post-Plucking Telogen > Anagen
B) Microarray mRNA expression
profiling of Sox4wt vs Sox4cKO
after plucking . Differentially
Expressed Genes (DEGs) include
known Sox4 targets)
C) PCR confirmation of microarray
E) Most DEGs specific to Telogen
vs. Anagen difference (stimulated
by plucking)
Gene Set Enrichment Analysis
compares results from other gene
expression studies, revealed
enrichment of genes for
cell cycle regulation, DNA repair,
and Wnt/β catenin signaling
F) Heat maps of top 20 DEGs in
these pathways in Soxwt vs SoxcKO
G) FDR(False Discovery Rate):
Likelihood that similarity between
sets is random
Induced Proliferation
Fig. 6 Sox4 is Required for
Oncogenic Transformation
A) Sox4lox/lox MEFs are resistant
to oncogenic transformation by
activated Ras (RasG12V)
B-D) DMBA/TPA mutagenesis:
Sox4cKO resistant to skin tumor
formation relative to wt (and
size of tumors formed smaller)
E) Sox4cKO/- and Sox4lox/lox
(partial loss of function)
also resistant to skin tumor
formation relative to wt (and
size of tumors formed smaller)
Cells recovered from each genotype
at time prior to tumor formation had
less proliferation markers (Ki67 &
P-H3) in SoxcKO relative to wt
Discussion: What did we learn from the study?
But also needed for
oncogenic transformation
Stress
Proliferation
Sox4 needed to maintain
stem cell populations
for skin tissue homeostasis &
regeneration
Delicate Balance
Required
Are there any caveats to the conclusions? What new or remaining questions are there?