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

Fig. 7.1

Epidermis:Interfollicular Epidermis (IFE)Sebaceous Glands (SG)Hair Follicles (HF)

Dividing Cells:Hair Follicle Stem Cells (HFSC)in Hair Bulge and HG

Dermis secretes signalingmolecules that regulate HFSC proliferation, differentiation, and self-renewalthrough Wnt/β catenin pathwayand Sox4 transcription factor

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

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.

Sox4

Sox4lox/lox

Skin HFSC or HG cell

Sox4cKO

in skinprogenitorcell

How were the Sox4lox/lox and Cre Transgenic Mice Made?

Fig. 18.49

Remove ES cells from Blastocystand culture in vitroAdd DNA w/

selectable marker(inserts intogenome in 1/104 ES cells) Inject transfected

ES cells into Blastocystand implant in mouse

Blastocysts w/transfected EScells develop intoadults, somecarrying transfectedDNA in GERMLINE

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 DisplayCancer Resistance & AcceleratedAging

Question: Phenotype in Sox4lox/lox

mice (knock-down intermediate)?

YESA) Strategy for making Sox4lox/lox

B) PCR confirmation that it was madeC) Sox4lox/lox smaller in sizeD) Sox4lox/lox smaller in weight

I) Sox4lox/lox has lower spontaneous cancer rate

E) Sox4lox/lox has lower survival in wks and Humane End PointF) Sox4lox/lox has lower Bone DensityG) Sox4lox/lox has higher Hernia RateH) Sox4lox/lox has lower mean telomere length in blood cells

J) 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) Normal hair coat appearance B) Sox4 expression absent in SoxcKO miceC) Expression of skin-specific differentiation genes normalD) Telomeres longer in HFSCs in Bulge of Sox4cKO mice (reduced replicative history)E) 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 9B) and Sox 4C) Hair regeneration reduced in Sox4cKO relative to Sox4wt

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 fromSox4lox/lox or Sox4cKO mice

J) Reduced wound healing inSoxcKO mice

Fig. 4 Sox4 is Required forNormal HFSC Activation duringHair Regeneration andWound Healing

Induced Proliferation

Fig. 5 Sox 4 is Required forInduction of Proliferative andDifferentiation Pathways duringPost-Plucking Telogen > Anagen

B) Microarray mRNA expressionprofiling of Sox4wt vs Sox4cKO

after plucking . Differentially Expressed Genes (DEGs) include known Sox4 targets)C) PCR confirmation of microarrayE) Most DEGs specific to Telogenvs. Anagen difference (stimulatedby plucking)Gene Set Enrichment Analysiscompares results from other gene expression studies, revealed enrichment of genes for cell cycle regulation, DNA repair, and Wnt/β catenin signalingF) 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

A) Sox4lox/lox MEFs are resistantto oncogenic transformation byactivated Ras (RasG12V)

B-D) DMBA/TPA mutagenesis:Sox4cKO resistant to skin tumorformation relative to wt (andsize of tumors formed smaller)

E) Sox4cKO/- and Sox4lox/lox

(partial loss of function) also resistant to skin tumorformation relative to wt (andsize of tumors formed smaller)

Fig. 6 Sox4 is Required for Oncogenic Transformation

Cells recovered from each genotypeat time prior to tumor formation hadless proliferation markers (Ki67 &P-H3) in SoxcKO relative to wt

Discussion: What did we learn from the study?

Stress Proliferation

Sox4 needed to maintainstem cell populationsfor skin tissue homeostasis &regeneration

But also needed foroncogenic transformation

Delicate BalanceRequired

Are there any caveats to the conclusions? What new or remaining questions are there?