Research ArticleCancer

The protein kinase p38α destabilizes p63 to limit epidermal stem cell frequency and tumorigenic potential

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Science Signaling  09 Oct 2018:
Vol. 11, Issue 551, eaau0727
DOI: 10.1126/scisignal.aau0727
  • Fig. 1 Loss of p38α signaling correlates with enhanced tumorigenesis in human barrier tissues.

    (A to C) Differential survival rates of the indicated TCGA patient groups with high and low p-p38 signals in their tumors are shown as P values by log-rank test. The cancer types are indicated by TCGA abbreviations, including LUSC (lung SCC) and ESCA (esophageal carcinoma). The survival of patients with LUSC and ESCA (n = 160 and 63 per group, respectively) is shown in Kaplan-Meier plots (B and C). (D and E) Human AK skin and SCC tumor sections (n = 10 and 16, respectively) were analyzed by immunostaining (D). Dotted line, the epidermal-dermal boundary (D). p-p38 signals were graded from 0 to 3 based on immunofluorescence (IF) intensity per cell (E). P values are obtained by Mann-Whitney test.

  • Fig. 2 Loss of p38α signaling results in enhanced skin tumorigenesis in mice.

    (A and B) Mice (n = 23) were subjected to DMBA-TPA skin tumorigenesis. Tumor incidence (A) and multiplicity (B) were determined over the indicated period. P values are obtained by log-rank test (A) and two-tailed unpaired Student’s t test (B). (C to G) DMBA-TPA–induced tumor sections from mice were analyzed by immunostaining/counterstaining for the indicated molecules (C, D, F, and G) and by hematoxylin and eosin (H&E) staining (E). Red arrowheads, peritumoral vasculature (E). Dotted lines, the tumor-stroma boundary (F and G). Images are representative of three to five tissue sections.

  • Fig. 3 Loss of p38α signaling results in enhanced epidermal regeneration after punch-biopsy wounding.

    (A to E) Mice (n = 12) were subjected to punch-biopsy wounding in the dorsal skin. Wound closure rates were determined over the indicated period and are shown in box-and-whisker plots (A). Wounds were photographed immediately (d0) and 3 days (d3) after wounding (B). Wound skin sections prepared 1 day after wounding were analyzed by H&E staining (C and D). Red arrowheads, wound margin; orange arrows, leading edge of regenerating epidermis (C and D). The extent of re-epithelialization 1 day after wounding was quantified on the basis of the distance between wound margin and epidermal leading edge and is shown as means ± SD (E). P values are obtained by two-tailed unpaired Student’s t test (A and E). Images are representative of five tissue sections.

  • Fig. 4 p38α restricts the frequency of keratinocytes with stem cell properties.

    (A and B) Epidermal cells from adult mice were plated and grown for colony formation. Colonies stained with crystal violet were photographed (A) and quantified from three experiments (B). P values are obtained by two-tailed unpaired Student’s t test. (C and D) Steady-state epidermal cells from adult mice (n = 6 per group) were incubated with Hoechst 33342. Label-refractory side populations (encircled in pink line) were detected by flow cytometry (C). The percentage of the epidermal side population (SP) was quantified and is shown as individual values and means ± SD (D). P values by two-tailed unpaired Student’s t test. (E and F) Steady-state skin sections (n = 13 to 14 per group) from adult mice were analyzed by immunostaining/counterstaining for the indicated molecules 15 days after 5-bromo-2′-deoxyuridine (BrdU) injection (E). White arrowheads, BrdU label-retaining cells (E). Epidermal label-retaining cell (LRC) frequency was determined on the basis of label-retaining cell numbers per image field and is shown as individual values and medians (F). P values were determined using Mann-Whitney test.

  • Fig. 5 p38α activity reduces p63 protein amounts and p63+ cell numbers in the epidermis and epidermal-derived tumors.

    (A to D) Mouse (A to C) and human (D) keratinocytes were left unstimulated or stimulated with TPA (100 nM), UVB (75 mJ/cm2), and anisomycin (10 μg/ml). Whole-cell lysates were prepared after the indicated durations of stimulation and analyzed by immunoblotting. Cells were preincubated with 5Z-7-oxozeaenol (Oz), SC409 (SC), SB202190 (SB), and D-JNKi (DJi) before stimulation (D). Blots are representative of three experiments. IRF6, interferon regulatory factor 6. (E to G) Steady-state (“none”) and TPA-treated skin sections (E), DMBA-TPA–induced tumor sections (F), and wound skin sections (G) from mice were analyzed by immunostaining/counterstaining for the indicated molecules. Solid and dotted lines, epidermal margins and the epidermal-dermal boundary, respectively (E). Red arrowheads, wound margin; orange arrows, leading edge of regenerating epidermis; solid and dotted lines, margins of eschar (e) and granulation tissue (g), respectively (G). Images are representative of five to seven tissue sections. (H) Human AK skin sections were analyzed by immunostaining for the indicated molecules. Red and white arrowheads, epidermal cell layers with differential immunofluorescence signals; dotted lines, the epidermal-dermal boundary; *Nonspecific staining. Images are representative of three tissue sections.

  • Fig. 6 p38α phosphorylates and destabilizes p63.

    (A) 32P-radiolabel p63 from in vitro kinase reactions was analyzed by autoradiography (AR) and immunoblotting (IB). Autoradiographic images and blots are representative of two experiments. GST, glutathione S-transferase. (B and C) 293T cells were transfected with plasmids expressing the indicated proteins. Whole-cell lysates prepared 24 hours after transfection were analyzed by immunoblotting; where indicated, cells were incubated with MG132 (5 μM) for 12 hours before lysate preparation (B). p63 was immunoprecipitated from transfected/MG132-treated cell lysates and analyzed by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie Blue staining (C). Solid and open circles, plasmid-expressed and endogenous p38α, respectively (B); arrow and dash, putative phosphorylated and unphosphorylated p63, respectively (B and C). Blots and gel images are representative of two experiments. HA, hemagglutinin. (D) Phosphorylation sites on ΔNp63α are depicted. Black and red arrows, putative and experimentally determined p38α phosphorylation sites, respectively. (E and F) KERT cells were infected with lentiviruses expressing the indicated p63 mutant derivatives. Whole-cell lysates prepared 48 hours after lentiviral infection were analyzed by immunoblotting; cells were incubated with cycloheximide (CHX; 20 μg/ml) for the indicated durations before lysate preparation (E). p63 amounts were quantified by densitometry (F). Blots are representative of three experiments. (G) 293T cells were transfected with plasmids expressing the indicated proteins. Whole-cell lysates prepared 24 hours after transfection were analyzed by immunoblotting. Solid and open circles, plasmid-expressed and endogenous p38α, respectively. Blots are representative of three experiments. (H and I) Human AK skin and skin SCC tumor sections (n = 10 and 20, respectively) were analyzed by immunostaining for the indicated molecules (H). Red and white arrowheads, epidermal cell layers with differential immunofluorescence signals; dotted line, the epidermal-dermal boundary; *Nonspecific staining (H). p-p63 signals were graded from 0 to 3 based on immunofluorescence intensity per cell (I). P values were determined by Mann-Whitney test.

  • Fig. 7 p38α reverses p63-mediated repression of tumor suppressor gene expression.

    (A) Mouse keratinocytes were left unstimulated or stimulated with UVB (75 mJ/cm2). RNA prepared 4 hours after stimulation was analyzed using DNA microarrays. (B) DMBA-TPA–induced tumor sections from mice were analyzed by immunostaining/counterstaining for the indicated molecules. *Nonspecific staining. Images are representative of five tissue sections. (C) Mouse keratinocytes were infected with control (C) or p63 shRNA-expressing (#1 and #2) lentiviruses and left unstimulated or stimulated with TPA (100 nM). RNA prepared 4 hours after stimulation was analyzed by quantitative polymerase chain reaction (qPCR). Data are means ± SD of three biological replicates. (D and E) Mice (n = 6) were subjected to DMBA-TPA skin tumorigenesis. Tumor incidence (D) and multiplicity (E) were determined over the indicated period. P values were obtained by log-rank test (A) and two-tailed unpaired Student’s t test (B).

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/551/eaau0727/DC1

    Fig. S1. Loss of p38α signaling does not enhance TPA-induced skin inflammation.

    Fig. S2. Refractoriness of epidermal side population cells to dye labeling depends on verapamil-sensitive membrane transporters.

    Fig. S3. Loss of p38α signaling does not reduce p63 mRNA amounts in epidermal keratinocytes and epidermal-derived tumors.

    Fig. S4. p63 mRNA and protein amounts vary in different types of human cancer and among different human cancer cell lines.

    Fig. S5. p38α phosphorylates p63 in vitro.

    Fig. S6. p38α is required for inducing or attenuating specific target genes in keratinocytes.

    Fig. S7. p63 represses MMP13 expression in keratinocytes.

    Table S1. LC-MS/MS detection of ΔNp63α-derived phosphopeptides containing p38 phosphoacceptor sites.

    Table S2. Oligonucleotide primers used in real-time qPCR.

  • This PDF file includes:

    • Fig. S1. Loss of p38α signaling does not enhance TPA-induced skin inflammation.
    • Fig. S2. Refractoriness of epidermal side population cells to dye labeling depends on verapamil-sensitive membrane transporters.
    • Fig. S3. Loss of p38α signaling does not reduce p63 mRNA amounts in epidermal keratinocytes and epidermal-derived tumors.
    • Fig. S4. p63 mRNA and protein amounts vary in different types of human cancer and among different human cancer cell lines.
    • Fig. S5. p38α phosphorylates p63 in vitro.
    • Fig. S6. p38α is required for inducing or attenuating specific target genes in keratinocytes.
    • Fig. S7. p63 represses MMP13 expression in keratinocytes.
    • Table S1. LC-MS/MS detection of ΔNp63α-derived phosphopeptides containing p38 phosphoacceptor sites.
    • Table S2. Oligonucleotide primers used in real-time qPCR.

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