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. 2018 Nov;20(11):1256-1266.
doi: 10.1038/s41556-018-0218-9. Epub 2018 Oct 22.

Oncogenic Activation of PI3K Induces Progenitor Cell Differentiation to Suppress Epidermal Growth

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Free PMC article

Oncogenic Activation of PI3K Induces Progenitor Cell Differentiation to Suppress Epidermal Growth

Zhe Ying et al. Nat Cell Biol. .
Free PMC article

Abstract

Oncogenic lesions are surprisingly common in morphologically and functionally normal human skin. However, the cellular and molecular mechanisms that suppress their cancer-driving potential to maintain tissue homeostasis are unknown. By employing assays for the direct and quantitative assessment of cell fate choices in vivo, we show that oncogenic activation of PI3K-AKT, the most commonly activated oncogenic pathway in cancer, promotes the differentiation and cell cycle exit of epidermal progenitors. As a result, oncogenic PI3K-AKT-activated epidermis exhibits a growth disadvantage even though its cells are more proliferative. We then sought to uncover the underlying mechanism behind oncogene-induced differentiation via a series of genetic screens in vivo. An AKT substrate, SH3RF1, is identified as a specific promoter of epidermal differentiation that has no effect on proliferation. Our study provides evidence for a direct, cell autonomous mechanism that can suppresses progenitor cell renewal and block clonal expansion of epidermal cells bearing a common and activating mutation in Pik3ca.

Conflict of interest statement

All authors provided intellectual input, vetted and approved the final manuscript, and declare that they have no financial and non-financial competing interests.

Figures

Fig. 1.
Fig. 1.. Oncogenic activation of the catalytic subunit of PI3K inhibits clonal expansion
a, Schematic of a genetic screen for regulators of clonal expansion, based on enrichment (orange) or depletion (blue) of individual barcoded gene-targeting lentivirus over time. b, Needle plot shows enrichment of growth promoting (orange) and growth inhibiting (blue) lentivirus in P21 epidermis relative to their initial abundance in the lentivirus pool (t=0). shScram and GFP are control constructs unchanged between t=0 and P21. Fold change shown is an average value from 3 biological replicates. c, Cre reporter mouse exchanges membrane-associated red (mT) fluorophore with green (mG) following Cre expression. Cre recombinase is expressed from a lentivirus that co-expresses PIK3CAH1047R ORF. d, Representative image of wild-type (LV-Cre) and oncogenic Pik3ca (LV-Cre-PIK3CAH1047R) clones in P21 epidermis of Cre-reporter (R26mT/mG) mice. Untransduced epidermis (red) surrounds transduced (green) cell clone. Scale bar, 100 μm. e, Quantification of cell numbers per clone in P21 epidermis demonstrates that oncogenic PI3K activation results in significant reduction in clone size relative to wild-type. Statistics were derived from n=91 WT and 90 PIK3CAH1047R clones pooled from 3 animals of each condition. Two-tailed t test, P-value<0.0001. Error bar: SD, center value: mean. Statistical source data for e are shown in Supplementary Table 4.
Fig. 2.
Fig. 2.. Oncogenic PI3K activation promotes epidermal differentiation but not apoptosis or senescence
a, Schematic of Pik3caH1047R/H1047R knock-in mouse. b, Quantifications of the rate of cell division, measured by EdU incorporation and average division interval (see Supplementary Fig. 2d and Methods), show that basal progenitors in Pik3ca 2X epidermis divide faster than wild-type (WT) at P21. Statistics based on n=3 animals of each condition. Two-tailed t test, P-value as indicated. Error bar: SD, center value: mean. c, Quantification of activated Caspase3 staining shows no significant difference between Pik3ca 2X and WT epidermis at P21. Statistics based on n=3 animals of each condition. Two-tailed t test, P-value as indicated. Error bar: SD, center value: mean. d, Senescence associated β-gal (SA-βgal) staining and Cdkn2a expression failed to detect any senescent cells in WT and Pik3ca 2X epidermis at P21. Statistics based on n=3 animals of each condition. Two-tailed t test, P-value as indicated. Error bar: SD, center value: mean. ND: not detected. e, Schematic of the EdU-BrdU pulse-chase differentiation assay. EdU only progeny cells (red), labeled by the initial two-hour pulse of EdU, are assessed for expression (brown) or absence (blue) of differentiation marker K10, six hours after completing the S-phase. Rate of renewal is expressed as the fraction of new K10 negative progenitors out of the total progeny. f, Oncogenic Pik3ca promotes differentiation in epidermis at embryonic, perinatal, and adult stages. Statistics based on n=3 animals of each condition. Error bar: SD, center value: mean. g-i GSEA (g) shows that oncogenic Pik3ca biases basal progenitor cell transcriptome towards differentiation. ES, NES, FDR generated by GSEA2 program. Gene ontology enrichment (h) in genes up- and down-regulated in Pik3ca 2X basal cells. Enrichment P-value generated by PANTHER. Expression of self-renewal and differentiation markers in WT and Pik3ca 2X basal progenitors (i). Statistics are based on RNA-seq performed in n=3 animals of each condition. g-i represent data pooled from 3 RNA-seq replicates. Statistical source data for b-d,f are shown in Supplementary Table 4.
Fig. 3.
Fig. 3.. Oncogenic PI3K/AKT induces differentiation via suppression of progenitor self-renewal
a, Mouse for epidermis-specific expression of photoactivatable mCherry transgene is used in conjunction with a lentivirus for expression of membrane-associated GFP and Cre recombinase. b, Schematic of an intravital imaging experiment to label individual epidermal progenitors by photo-activating H2B-PAmCherry, and follow them as they divide, and their daughter cells commit to renewing or differentiating fate choices. c, Membrane-associated GFP (green) that marks the epidermal field is co-expressed with Cre-recombinase in a lentivirus (LV-Cre-mGFP). The progeny of the photo-activated basal cell (PA) are marked by Roman numerals (I, II). Their fate is reported by morphology, distance from the basement membrane, and mitotic potential (a, b indicate 2nd generation progeny). Scale bar, 25 μm. d, Symmetric renewal is significantly suppressed in ubiquitously activated Pik3ca 2X epidermis. Statistics based on n=3 animals of each condition. 96 wild-type and 102 Pik3ca 2X divisions were scored. Two-tailed t test, P-value as indicated. Error bar: SD, center value: mean. e and f, Schematic (e) and representative image (f) of live imaging of single CreER-transduced mT/mG cells activated by tamoxifen at P19 produced three modes of fate choice 48hrs post activation. Scale bar, 25 μm. g, Progenitor cell renewal is significantly suppressed in activated Pik3ca 2X epidermis. Statistics based on n=3 animals of each condition. 296 wild-type and 317 Pik3ca 2X doublets were scored. Two-tailed t test, P-value as indicated. Error bar: SD, center value: mean. Statistical source data for d,g are shown in Supplementary Table 4.
Fig. 4.
Fig. 4.. Oncogenic PI3K activation results in long-term clonal loss and suppresses tumor initiation
a, Cre-inducible expression of oncogenic Pik3caH1047R is achieved in cells transduced with lentivirus for Tamoxifen (TAM)-dependent activation of Cre recombinase. Skin epidermis treated with TAM at postnatal (P) day 19 was imaged at P20, 21, 35, 42 and P105 to quantify clone size in wild-type and Pik3ca 2X epidermis. Pik3ca 2X clones are significantly smaller then wild-type at P35 and P42, and are lost from the tissue by P105. Statistics are based on n=3 animals of each condition, clone numbers for each day/condition: WT (P20:101, P21:84, P28:134, P35:129, P42:135, P105:93); Pik3ca 2X (P20:142, P21:127, P28:170, P35:88, P42:89). Two-tailed t test, P-value as indicated. Box 10–90 percentile, error bar: SD, center value: mean. b, Representative image of wild-type (WT) tissue and Pik3ca 2X clones in P105 epidermis. H2B-GFP (green nuclear staining) was used as transduction control. Scale bar, 100 μm. 90 WT regions and 93 Pik3ca 2X clones were imaged. c, Oncogenic Pik3ca suppresses rate of renewal in HPV E7 oncogene transduced epidermis and tumor. Statistics based on n=3 animals of each condition. Two-tailed t test, P-value as indicated. Error bar: SD, center value: mean. d and e, Representative images (d) and statistics (e) show that oncogenic Pik3ca suppresses clonal expansion driven by HPV E7 oncogene. Statistics were derived from 36 WT+LV-HPV-E7 and 28 Pik3ca 2X+LV-HPV-E7 clones pooled from 3 animals of each condition. Two-tailed t test, P-value as indicated. Error bar: SD, center value: mean. Scale bar, 100 μm. f, Kaplan–Meier survival curve indicates oncogenic Pik3ca significantly delays HPV E7 driven tumor initiation. Statistics are based on n=8 animals of each condition. Log-rank test, P-value=0.0002. Statistical source data for a,c,e are shown in Supplementary Table 4.
Fig. 5.
Fig. 5.. Genetic screens identify SH3RF1 as a specific mediator of oncogenic PI3K/AKT-induced differentiation but not proliferation
a, Schematic of in vivo genetic screens for regulators of progenitor differentiation and proliferation among 242 known AKT substrates. Assays are based on enrichment (orange) or depletion (blue) of shRNAs in a6-Integrinhigh basal progenitors relative to differentiated suprabasal a6-Integrinlow cells (differentiation screen) or non-dividing EdU- relative to dividing EdU+ epidermal progenitors (proliferation screen). b-d, In vivo differentiation (b) and proliferation (c) screens identify Sh3rf1 as the top-ranking promoter of progenitor renewal that does not affect cell division (d). Fold change is an average value from n=3 biological replicates of each genetic screen. Significance quantified by Deseq2, with cutoff P-value=0.01. e and f, Immunoprecipitation of endogenous (e) and V5-tagged SH3RF1 (f) from epidermal lysates followed by Western blotting using phosphor-AKT-substrate specific antibody shows that SH3RF1 is phosphorylated by AKT in epidermis, and that its phosphorylation status is enhanced in Pik3ca 2X tissue. Experiment was repeated 3 times independently with similar results. Unprocessed blots see Supplementary Fig. 8. Depletion of Sh3rf1 in wild-type epidermis promotes progenitor differentiation without an effect on cell division rate, n=3 animals of each condition. Two-tailed t test, P-value as indicated. Error bar: SD, center value: mean. h, Overexpression of Sh3rf1 inhibits oncogenic Pik3ca-driven differentiation (left) without an effect on the rate of cell division (right, n=3 animals of each condition). Two-tailed t test, P-value as indicated. Error bar: SD, center value: mean. i and j, Representative image (i) and quantification (j) shows that overexpression of SH3RF1 promotes clone growth in wild-type and rescues clone loss following oncogenic PI3K activation. Statistics were derived from n=90 clones pooled from 3 animals of each condition. Two-tailed t test, P-value as indicated. Error bar: SD, center value: mean. Scale bar, 100 μm. Statistical source data for g,h,j are shown in Supplementary Table 4.
Fig. 6.
Fig. 6.. AKT-mediated phosphorylation of SH3RF1 is required for oncogenic Pik3ca-induced differentiation and suppression of clonal growth
a, Schematic of SH3RF1 mutation constructs: deletion of Ring domain, SH3RF1ΔRing; AKT phosphorylation site mutant, SH3RF1SA; AKT phosphorylation mimetic mutant, SH3RF1SD. b, Immunoprecipitation of V5-tagged SH3RF1 mutants from transduced epidermis lysate followed by Western blotting using phosphor-AKT-substrate specific antibody confirmed that S304 is phosphorylated by AKT in epidermis. Experiment was repeated in n=3 biological replicates with similar results. Two-tailed t test, P-value as indicated. Error bar: SD, center value: mean. Unprocessed blots see Supplementary Fig. 8. c, EdU-BrdU pulse-chase differentiation assay shows that SH3RF1ΔRing was equal to SH3RF1WT in promoting renewal, and that SH3RF1SA further enhances renewal while SH3RF1SD does not, in both wild-type and Pik3ca 2X epidermis. Statistics are based on n=3 animals of each condition. Two-tailed t test, P-value as indicated. Error bar: SD, center value: mean. d and e, Representative image (d) and quantification (e) shows that SH3RF1ΔRing was no different from SH3RF1WT in promoting clonal growth, and that SH3RF1SA but not SH3RF1SD further enhance clonal growth, in both wild-type and Pik3ca 2X epidermis. Statistics are based on 90 clones from n=3 animals of each condition. Two-tailed t test, P-value as indicated. Error bar: SD, center value: mean. Scale bar, 100 μm. Statistical source data for b,c,e are shown in Supplementary Table 4.
Fig. 7.
Fig. 7.. SH3RF1 is a scaffold for JNK signaling in oncogenic Pik3ca-driven differentiation
a, Model of how AKT signaling can promote epidermal progenitor cell differentiation. b, Transcriptome of Pik3ca 2X progenitors shows significant suppression of JNK signature genes. ES, NES, FDR generated by GSEA2 program. Statistics are based on RNA-seq performed in n=3 animals of each condition. c, p-JNK staining in indicated samples, dashed line indicates basement membrane. Scale bar, 25 μm. d, Western blotting and quantification of JNK phosphorylation in WT and Pik3ca 2X epidermis. Significant reduction of phospho-JNK signal in Pik3ca 2X epidermis is rescued by SH3RF1 expression. Experiment was repeated in n=3 biological replicates with similar results. Two-tailed t test, P-value as indicated. Error bar: SD, center value: mean. Unprocessed blots see Supplementary Fig. 8. e, SH3RF1ΔRing and SH3RF1WT but not SH3RF1SD rescue phospho-JNK suppression in Pik3ca 2X epidermis, while SH3RF1SA can further enhance it. Experiment was repeated in n=3 biological replicates with similar results. Two-tailed t test, P-value as indicated. Error bar: SD, center value: mean. Unprocessed blots see Supplementary Fig. 8. f and g, MLK1 rescues oncogenic Pik3ca-induced suppression of phospho-JNK (f) and differentiation (g). Experiment was repeated in n=3 biological replicates with similar results (f). Statistics are based on n=3 animals of each condition (g). Two-tailed t test, P-value as indicated. Error bar: SD, center value: mean. Unprocessed blots see Supplementary Fig. 8. h and i, Knockdown of Jnk1 blocks SH3RF1SA enhanced phospho-JNK (h) and progenitor renewal (i). Experiment was repeated in n=3 biological replicates with similar results (h). Statistics are based on n=3 animals of each condition (i). Two-tailed t test, P-value as indicated. Error bar: SD, center value: mean. Statistical source data for d-i are shown in Supplementary Table 4.

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