Figure 5. RAC1 and RAC2 are uniquely expressed in Tgfbr2-deficient transition zone tumors compared to DMBA-induced Tgfbr2-deficient skin SCC.

(A–D) Immunofluorescence staining with antibodies against RAC1, RAC2 and CD34 revealed strong expression of RAC1 and RAC2 in the anorectal SCC tumor compared to the backskin SCC. Skin tumors come from cKO mice treated for 16 weeks topically with the chemical mutagen 7,12-dimethyl-benz[a] anthracene (DMBA) as previously described (Guasch et al., 2007). Some RAC1-positive cells (A) and clusters of RAC2-positive tumor cells (C) correlate with CD34+ tumor cells (white arrows) in the anorectal SCC but not in the skin SCC (B, D). All images have been acquired using the same laser parameters and exposure time. DAPI counterstains nuclei in blue. Abbreviation: bv, blood vessel. Scale bars = 20 µm (A–A’’–B–B’’), 10 µm (C–C’’–D–D’’). n = 3 different skin and anorectal tumors tested for each antibody. See also Figure 5—figure supplement 1.

DOI: http://dx.doi.org/10.7554/eLife.22914.013

Figure 5—figure supplement 1. Venn diagrams of cell-type specific signatures in various skin SCC.

CD34+ and CD34− anorectal TZ SCC TGFβ-deficient signatures (Supplementary file 1) have been compared with all databases in Table 1 of Schober and Fuchs (2011) that are in the same genetic drivers Tgfbr2-deficient with and without loss of Focal Adhesion Kinase (FAK) using R program available through the Comprehensive R Archive Network. CD34+ and CD34− anorectal TZ SCC TGFβ-deficient signatures have been also compared with other transcriptional profiles of CSC from skin cancers in a TGFβ-intact background (DMBA/TPA treated) in a WT background (Schober and Fuchs, 2011) and VEGF gain of function (from Beck et al., 2011), https://www-ncbi-nlm-nih-gov.gate2.inist.fr/geo/query/acc.cgi?acc=GSE31465). The overlaps represent commonly enriched genes in corresponding populations compared to all others. In a TGFβ-deficient background, only six genes were found significantly commonly highly expressed between the Tgfbr2 cKO CD34+ SCC anorectal TZ cells and the Tgfbr2 cKO CD34High SCC skin cells (***p value=1.57×10-7), seven genes were found significantly commonly highly expressed between the Tgfbr2 cKO CD34+ SCC anorectal TZ cells and the Tgfbr2 cKO CD34Low SCC skin cells (***p value=7.48×10-5) and nine genes were found significantly commonly highly expressed between the Tgfbr2 cKO CD34+ SCC anorectal TZ cells and the Tgfbr2/FAK double KO CD34Low SCC skin cells (***p value=1.98×10-10). In a WT background, 11 genes were found common between the Tgfbr2 cKO CD34+ SCC anorectal TZ cells and the CD34Low SCC skin cells (***p value=1.49×10-11) and 21 genes when FAK is deficient (***p value=5.35×10-26). In skin SCC with a gain of function of VEGF (Beck et al., 2011), 45 genes upregulated in CD34+VEGF versus CD34+ skin SCC were found also significantly highly expressed in the Tgfbr2 cKO CD34+ SCC anorectal TZ cells (***p value=2.62×10-39) and 40 genes downregulated in CD34+VEGF versus CD34+ skin SCC were found significantly highly expressed in the Tgfbr2 cKO CD34+ SCC anorectal TZ cells (***p value=2.69×10-53). The genes have been listed and classified in five categories: ECM organization, epithelial to mesenchymal transition, adhesion/invasion/metastasis, Rac/Rho/Ras pathway and others. All p values have been calculated with the hypergeometric (HG) test and are represented in the graph. The orange vertical bar indicates the statistical significance at 5%.

DOI: http://dx.doi.org/10.7554/eLife.22914.014