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 signaling molecules that regulate HFSC proliferation, differentiation, and self-renewal through Wnt/β catenin pathway and 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 Sox4 lox/lox Skin HFSC or HG cell Sox4 cKO in skin progenitor cell

How were the Sox4 lox/lox and Cre Transgenic Mice Made? Fig Remove ES cells from Blastocyst and culture in vitro Add DNA w/ selectable marker (inserts into genome in 1/10 4 ES cells) Inject transfected ES cells into Blastocyst and implant in mouse Blastocysts w/ transfected ES cells develop into adults, some carrying transfected DNA 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 Sox4 lox/lox Mice Display Cancer Resistance & Accelerated Aging Question: Phenotype in Sox4 lox/lox mice (knock-down intermediate)? YES A)Strategy for making Sox4 lox/lox B)PCR confirmation that it was made C)Sox4 lox/lox smaller in size D)Sox4 lox/lox smaller in weight I) Sox4 lox/lox has lower spontaneous cancer rate E) Sox4 lox/lox has lower survival in wks and Humane End Point F)Sox4 lox/lox has lower Bone Density G)Sox4 lox/lox has higher Hernia Rate H)Sox4 lox/lox has lower mean telomere length in blood cells J) Reduced Sox4 expression in all tissues of Sox4 lox/lox (lox sites flanking gene must interfere with its transcription)

Fig. 2 Normal Skin Stratification in Sox4 cKO, but Bulge Cells Have Reduced Replicative History A)Normal hair coat appearance B)Sox4 expression absent in Sox cKO mice C)Expression of skin-specific differentiation genes normal D) Telomeres longer in HFSCs in Bulge of Sox4 cKO mice (reduced replicative history) E) 5% HFSC telomeres 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 Sox4 cKO relative to Sox4 wt

A-H) Decrease in proliferating cells as evidenced by decreased Ki67 (proliferation marker-rRNA txn) & increased γH2AX, p53 (cell cycle arrest markers) in Sox4 cKO I) Reduced number of large, differentiated colonies in cultured keratinocytes from Sox4 lox/lox or Sox4 cKO mice J) Reduced wound healing in Sox cKO mice Fig. 4 Sox4 is Required for Normal HFSC Activation during Hair Regeneration and Wound Healing

Induced Proliferation Fig. 5 Sox 4 is Required for Induction of Proliferative and Differentiation Pathways during Post-Plucking Telogen > Anagen B) Microarray mRNA expression profiling of Sox4 wt vs Sox4 cKO 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 Sox wt vs Sox cKO G) FDR(False Discovery Rate): Likelihood that similarity between sets is random

A) Sox4 lox/lox MEFs are resistant to oncogenic transformation by activated Ras (Ras G12V ) B-D) DMBA/TPA mutagenesis: Sox4 cKO resistant to skin tumor formation relative to wt (and size of tumors formed smaller) E) Sox4 cKO/- and Sox4 lox/lox (partial loss of function) also resistant to skin tumor formation relative to wt (and size of tumors formed smaller) Fig. 6 Sox4 is Required for Oncogenic Transformation Cells recovered from each genotype at time prior to tumor formation had less proliferation markers (Ki67 & P-H3) in Sox cKO relative to wt

Discussion: What did we learn from the study? Stress Proliferation Sox4 needed to maintain stem cell populations for skin tissue homeostasis & regeneration But also needed for oncogenic transformation Delicate Balance Required Are there any caveats to the conclusions? What new or remaining questions are there?