TCF7L1 promotes skin tumorigenesis independently of β-catenin through induction of LCN2
- Baylor College of Medicine, United States
- Michael E. DeBakey VA Medical Center, Baylor College of Medicine, United States
- University of Texas MD Anderson Cancer Center, United States
- Moffitt Cancer Center, United States
Figures
Figure 1
TCF7L1 is overexpressed in papillomas and skin SCC.
(A) Immunofluorescence analyses of frozen sections of normal skin, papilloma and SCC induced by DMBA/TPA using antibodies against TCF7L1 (green) and β4-integrin (red). (B) Immunohistochemistry of …
Figure 2 with 3 supplements
TCF7L1 overexpression increases tumor incidence and multiplicity and accelerates malignancy progression in the mouse model of skin SCC.
(A) Experimental scheme testing the role of TCF7L1 in tumorigenesis. Eight-week-old tet-inducible Tcf7l1 (KRT14-rtTA;TRE-Tcf7l1) and control (KRT14-rtTA or TRE-Tcf7l1) mice were put on a …
Figure 2—figure supplement 1
Prolonged induction of TCF7L1 in adult skin results in hyperproliferation.
(A–B) Immunofluorescence image of Ki-67 (green) and myc-TCF7L1 (red) expression in adult skins from control (KRT14-rtTA) or tet-inducible Tcf7l1 (KRT14-rtTA;TRE-Tcf7l1) mice that were placed on a …
Figure 2—figure supplement 2
Myc epitope-tagged TCF7L1 is induced in skins and tumors of tet-inducible mice on a doxycycline-containing diet.
Normal skins from control (KRT14-rtTA) or TCF7L1-induced (KRT14-rtTA;TRE-Tcf7l1) mice along with papilloma and SCC samples from TCF7L1-induced mice were analyzed by immunofluorescence with …
Figure 2—figure supplement 3
Tet-inducible TCF7L1 is tightly regulated and not sufficient to promote tumor formation.
(A–B) Eight-week-old tet-inducible TCF7L1 (KRT14-rtTA;TRE-Tcf7l1) and control (KRT14-rtTA or TRE-Tcf7l1) mice were maintained on a doxycycline-free diet and their backskins were treated with a high …
Figure 3 with 1 supplement
Downregulation of TCF7L1 and TCF7L2 reduces human skin SCC growth.
(A) Expression of TCF7L1 and TCF7L2 in adult human keratinocytes (KER) and different human SCC cell lines, including SCC12, SCC13, SRB1 and SRB12. The human skin SCC cell line SCC13 was transduced …
Figure 3—figure supplement 1
Silencing TCF7L1 and TCF7L2 in SCC cells reduces proliferation in vitro and in vivo.
(A) Silencing TCF7L1 and TCF7L2 reduces SCC cell proliferation in vitro. Human SCC (SCC13) cells expressing inducible shRNA against TCF7L1 and TCF7L2 (shTCF7L1#1+shTCF7L2#1) or nonspecific control …
Figure 4
TCF7L1 stimulates tumor growth and increases tumorigenic capacity.
(A) TCF7L1 promotes xenogafted tumor growth. Human skin SCC cell line SRB12 transduced with control vector or vector expressing tet-inducible Tcf7l1 were drug selected and expanded. 1 million cells …
Figure 5 with 1 supplement
TCF7L1 promotes tumor growth independently of β-catenin binding.
The human skin SCC cell line SCC13 was transduced with pINDUCER21 lentiviral vectors expressing GFP alone or GFP with tet-inducible, myc-tagged murine Tcf7l1 or Tcf7l1 deletion mutants. After …
Figure 5—figure supplement 1
Activity of TCF7L1, TCF7L1 deletion mutants and TCF7L2 on TCF/β-catenin responsive promoter (TOPFlash).
Mouse keratinocytes that were transduced and drug selected to express tet-inducible Tcf7l1, Tcf7l1 deletion mutants, or Tcf7l2 were transiently co-transfected with TOPFlash reporter plasmids and TK-R…
Figure 6 with 2 supplements
TCF7L1 suppresses HRASG12V-induced senescence independently of β-catenin interaction.
Mouse primary keratinocytes were transduced to express tet-inducible Tcf7l1 or Tcf7l1 deletion mutants. After cell line establishment by G418 selection, cells were re-infected to express oncogenic HR…
Figure 6—figure supplement 1
TCF7L1 overrides HRASG12V-induced senescence in fibroblasts.
(A) Measurement of SA-β-gal activity by X-gal staining at pH 6.0 in fibroblasts transduced to express oncogenic HRASG12V with or without TCF7L1 overexpression. Bar denotes 200 µm. (B) Quantification …
Figure 6—figure supplement 2
ΔNβcat has no effect on HRASG12V-induced senescence in keratinocytes.
Tcf7l1-inducible primary keratinocytes were transduced to express oncogenic HRASG12V and then re-transduced with pLenti-RFP or pLenti-RFP-ΔNβcat after two days. After drug selection and/or …
Figure 7
TCF7L1 promotes cell migration in SCC cells independently of β-catenin binding.
The human skin SCC cell lines SCC13 and SRB12 were transduced with tet-inducible Tcf7l1 or Tcf7l1 deletion mutants. After drug selection, the transduced cells were treated with doxycycline for 48 hr …
Figure 8
Overexpression of TCF7L2 promotes tumor growth, increases cell migration, and represses HRASG12V-induced senescence similarly to TCF7L1.
The human skin SCC cell line SCC13 was transduced to express tet-inducible, myc-tagged TCF7L1 or TCF7L2. After G418 drug selection, transduced cells were grafted into flanks of immunodeficient nude …
Figure 9
TCF7L1 overexpression alters transcriptional landscape in SCC cells.
The human skin SCC cell line SCC13 was transduced with lentiviral vectors expressing tet-inducible Tcf7l1 or Tcf7l1 deletion mutants. After drug selection, the transduced cells were treated with …
Figure 10 with 3 supplements
TCF7L1 promotes tumor growth through induction of LCN2.
(A) Real time PCR analysis of expression of LCN2 in SCC13 and SRB12 cells that were transduced to express Tcf7l1 and its various mutants. (B) Real time PCR analysis of Lcn2 in enriched epithelial …
Figure 10—figure supplement 1
LCN2 is induced by TCF7L1 and is overexpressed in murine skin papilloma and SCC.
(A) Immunofluorescence images of LCN2 staining in control and TCF7L1-induced skin. Control or tet-inducible Tcf7l1 mice were on a doxycycline containing diet for five days and their skins were …
Figure 10—figure supplement 2
TCF7L1 and LCN2 are expressed in human skin SCC.
(A) Representative images of negative and positive immunohistochemical staining of TCF7L1 and LCN2 in human skin SCC (middle and bottom panels) and adjacent normal skin (top). Bar denotes 100 µm. (B)…
Figure 10—figure supplement 3
Overexpression of TCF7L1 increases neutrophil infiltration independently of LCN2.
Xenografted tumors (n = 3) expressing empty vector or vector expressing tet-inducible Tcf7l1 with or without shLCN2#1 were dissociated and analyzed by flow cytometry for CD11b+Ly6G+Ly6C+ …
Additional files
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Supplementary file 1
Differentially-expressed gene lists and associated pathway analyses of human SCC (SCC13) cells overexpressing TCF7L1 or TCF7L1 deletion mutants.
Tab one shows the list of genes that are differentially expressed between SCC13 cells overexpressing TCF7L1 or TCF7L1 mutants and cells expressing control vector. For genes in the datasets, HUGO symbol, Cufflinks XLOC identifier and locus are indicated. In the differentially expressed gene list, fold changes were computed after anti-log2-transformation, and t statistics (p values and FDR-adjusted q values) were computed for the corresponding coefficient and the significance was determined by q < 0.05. Tab two shows the genes represented in the heat map. The heat map with unsupervised clustering was constructed with 500 genes that have the largest coefficient of variation based on batch-corrected FPKM counts (fragments per kilobase of transcript per million mapped reads), and scaled over geometric mean. The red and green indicate expression values above or below the mean expression, respectively. Tab three and four respectively show a list of GO term biological process and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that were derived from significantly induced or repressed gene lists with a twofold difference by the overexpression of TCF7L1 and TCF7L1ΔN but not TCF7L1*. Top 100 altered pathways were listed by p-value ranking.
- https://doi.org/10.7554/eLife.23242.022
