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.
Uncontrolled tissue expansion is the fundamental feature of cancer that is thought to be acquired early in tumorigenesis1. Oncogenes are thought to be the major drivers of growth in cancer. Oncogenes fuel tissue expansion by accelerating the cell cycle, inhibiting cell death or cell cycle arrest, and are believed to promote self-renewal of tissue stem cells1,2. Yet, skin epidermis, the tissue of origin for basal cell carcinoma and squamous cell carcinoma (SCC), was recently shown to contain an abundance of cells carrying activated oncogenes without any consequences. Moreover, clones containing activated oncogenes did not appear to be under positive selection, and clonal expansion was only associated with lesions in a small set of genes3. This surprising observation indicated that tumour-suppressive mechanisms are at work early on in epidermis to counteract growth-promoting functions of activated oncogenes and to keep clonal expansion under stringent control.
Senescence is a well-documented growth-suppressive mechanism by which cells respond to an oncogenic stress in a cell autonomous manner. Activation of BRAF V600E in nevi4 and NRAS in lymphocytes5 can induce cellular senescence in vivo. Similarly, loss of the tumour suppressor PTEN, which activates oncogenic PI3K–AKT signalling, induces senescence in prostate epithelium6. Apoptosis can also suppress oncogene-driven clonal expansion, as demonstrated following the overexpression of MYC and E1A in cell culture7,8,9. While highly efficient at restricting oncogene-driven growth, both senescence and apoptosis bring on a dramatic loss of cells and their growth potential. As such, they are not compatible with observations that skin epithelium maintains its structure, function and rapid turnover despite the massive amount of oncogenic lesions.
Growth in skin epithelium is driven by progenitor cells that can self-renew, to maintain the growth potential of tissue, and differentiate into postmitotic progeny, which provide the form and function of skin10,11. Although these cell fate decisions directly control the number of progenitors in a tissue over time, and are therefore a critical determinant of its growth potential1,12, whether they contribute to the regulation of clonal expansion in the context of oncogenic stress is not known.
The PI3K–AKT pathway is commonly hyperactivated in cancers13, and suppression of PI3K signalling has been shown to significantly inhibit proliferation and cell survival in epidermal SCC14,15. Yet, despite the observation that oncogenic mutations in the PI3K–AKT pathway are among the most common lesions in SCCs and that robust PI3K–AKT activity is also detected in premalignant epithelia16,17, very little is known about how they affect clonal expansion. In addition, the effect of normal PI3K–AKT signalling on epidermal stem cell renewal seems to be context-dependent. Activation of PI3K in organotypical cultures of epidermal progenitors was shown to promote colony formation and to decrease differentiation marker expression18,19, whereas in conventional culture conditions, it had the opposite effect20,21. In epidermal development, inhibition of PI3K–AKT signalling was shown to suppress the expression of the progenitor cell marker TP6322, while loss of PDK1, the upstream activator kinase of AKT, resulted in increased TP63 expression23. Despite the different effect on TP63 expression, both studies reported that suppression of PI3K–AKT signalling blocked epidermal stratification. In the adult, expression of myristoylation signal attached AKT was shown to promote the expansion of hair follicle stem cells24,25, supporting the long-standing idea that oncogenes drive stem cell renewal to contribute to tumorigenesis2. Importantly, the effect of oncogenic mutations in the PI3K–AKT pathway on epithelial progenitor cell renewal and differentiation in adult epidermis, where tumours usually originate, has never been directly tested.
In the current study, we show that oncogene-induced differentiation is the dominant growth-suppressive mechanism in oncogenic Pik3ca-activated epidermis that restricts clonal expansion. Using independent and direct measurements of cell fate choice in both fixed tissues and live animals, we show that oncogenic activation of PI3K in adult epidermis results in a cell autonomous suppression of symmetrical renewal that drives reduced clonal expansion and long-term loss of oncogene-expressing epithelial cells. We employ a series of genetic screens in vivo and uncover an AKT substrate, SH3RF1, as a specific promoter of progenitor renewal that has no effect on proliferation. Finally, we use tests of genetic epistasis in vivo to show that oncogenic activation of PI3K signalling results in AKT-mediated suppression of SH3RF1 scaffold function in supporting pro-renewal JNK signalling.
Oncogenic activation in the PI3K pathway inhibits clonal expansion
We selected 35 known drivers of SCCs3 (Supplementary Fig. 1a–c) and generated open-reading frame (ORF)- and short hairpin RNA (shRNA)-expressing constructs to model their gain- and loss-of-function lesions. To test how these cancer drivers affect epithelial growth, we introduced them as a lentiviral pool into mouse epidermis via ultrasound-guided in utero microinjection26,27. We reasoned that constructs that affect growth would become enriched or depleted in the epidermis over time, which we could measure by sequencing27 (Fig. 1a). Consistent with previous findings, lesions associated with clonal expansion in human skin3 were among the top promoters of epidermal growth (Fig. 1b; Supplementary Table 1). Unexpectedly, our screen also identified several oncogenes in the PI3K–AKT pathway as significant suppressors of growth (Fig. 1b). We focused on Pik3ca, which encodes the catalytic subunit of PI3K and is the most commonly mutated proto-oncogene across epithelial cancers13,28,29 (Supplementary Fig. 1d). To independently measure the impact of oncogenic Pik3ca on epithelial growth, we transduced epidermis of Cre-reporter mice (R26mT/mG)30 with lentivirus expressing Cre-recombinase (LV-Cre) alone or together with oncogenic Pik3ca (Pik3caH1047R) at clonal density. We observed that oncogenic Pik3ca suppressed epidermal growth and clonal expansion, thus validating our genetic screen (Fig. 1c–e).
PI3K activation promotes the differentiation of epidermal progenitor cells
To probe how oncogenic Pik3ca inhibits clonal expansion, we used a conditional knock-in mouse31,32,33 in which activated Pik3ca is expressed at physiological levels and under endogenous control (Fig. 2a). We established that Cre-recombinase efficiently replaces the wild-type protein with its oncogenic form, and results in the activation of AKT in Pik3caH1047R/H1047R (termed Pik3ca 2× from hereon) epidermis (Supplementary Fig. 2a,b). We next measured progenitor cell proliferation and observed a significant approximately twofold increase in Pik3ca 2× tissues compared with wild-type during perinatal and adult stages (Fig. 2b; Supplementary Fig. 2c,d). We also tested for apoptosis and senescence, but detected no change in either of these events (Fig. 2c,d). Increased proliferation not balanced by apoptosis or senescence stood in contrast to our observation that oncogenic Pik3ca inhibited clonal growth, and suggested that there were additional mechanisms of growth inhibition.
Proliferation in wild-type and Pik3ca 2× epidermis was restricted to keratin 10 (K10)-negative basal progenitors (Supplementary Fig. 3a,b). We reasoned that an increase in their differentiation into post-mitotic progeny would contract the proliferative compartment of tissue and therefore suppress growth. To test whether oncogenic Pik3ca affects differentiation, we developed an assay of progenitor renewal based on 5-ethynyl-2′-deoxyuridine (EdU) and 5-bromodeoxyuridine (BrdU) pulse–chase incorporation (Fig. 2e). We established that a 2-h EdU pulse followed by a 6-h BrdU chase enabled >95% of EdU-positive-only progenitors to complete mitosis (~3% remain in G2). This protocol also enabled the tracing of their daughter cell fate based on the expression of the differentiation marker K10 in both wild-type and Pik3ca 2× epidermis (Supplementary Fig. 3c,d). We readily detected EdU-positive-only cells and their differentiation status, and used the fraction of K10-negative progenitors in the EdU-positive-only daughter cell population as a measure of progenitor cell renewal rate in vivo (Fig. 2e; Supplementary Fig. 3e).
Using EdU–BrdU pulse–chase assays, we first measured the progenitor renewal rate in wild-type epidermis and found that it ranges from ~0.75 at birth to ~0.5 in the adult (postnatal day 21 (P21) to P84; Fig. 2f). Strikingly, we observed a significant decrease in progenitor renewal in Pik3ca 2× epidermis across all stages (Fig. 2f), suggesting that the expression of oncogenic Pik3ca promotes differentiation. Importantly, increased differentiation was independent of cell crowding (Supplementary Fig. 4a) and of whether oncogenic Pik3ca was expressed clonally or throughout the epithelium, suggesting a cell autonomous mechanism (Supplementary Fig. 4b,c). In addition, differentiation marker expression and tissue turnover rates were similar in control and in Pik3ca 2× epidermis (Supplementary Fig. 4d,e), indicating that oncogenic Pik3ca promotes differentiation by regulating the initial cell fate choice. Interestingly, we found that the renewal rate of wild-type progenitors adjacent to Pik3ca 2× clones (≤5 cells from Pik3ca 2× clones) was elevated (Supplementary Fig. 4b,c). This was not seen in cells that were nearby but not adjacent to Pik3ca 2× clones, suggesting that the local increase in the renewal of wild-type progenitors could balance the loss of oncogene-expressing progenitors (Supplementary Fig. 4a–c).
Next, we performed RNA sequencing (RNA-seq) on basal progenitors expressing integrin α6 (α6 Itghigh), isolated by fluorescence-activated cell sorting (FACS), from wild-type and Pik3ca 2× epidermis. We found that oncogenic Pik3ca shifts the progenitor expression profile towards differentiation in both mouse34,35 and human36 (Fig. 2g; Supplementary Fig. 4f). Gene ontology analysis further revealed an increased expression of cell cycle regulators and downregulation of negative regulators of differentiation in basal progenitors in Pik3ca 2× epidermis (Fig. 2h). The increased differentiation in Pik3ca 2× progenitors was also evidenced by the expression of differentiation markers and regulators, including the suppression of Id237 and Sox938 and the elevation of Notch1, Notch339, Krt1 and Krt1040 (Fig. 2i). Together, our data demonstrate that oncogenic Pik3ca promotes the increased differentiation and cell cycle exit of progenitor cells in the epidermis.
PI3K activation directly modifies cell fate choice in epidermis to inhibit renewal
In epidermis, cell fate choice is associated with progenitor cell divisions, which give rise to two mitotically active daughters (symmetric renewal), two post-mitotic daughters (symmetric differentiation) or divide asymmetrically to maintain one progenitor cell and generate one differentiated daughter. To probe whether oncogenic Pik3ca promotes differentiation by regulating progenitor cell fate choice, we used two-photon intravital microscopy (Supplementary Fig. 5a). To visualize dividing progenitors and to follow the fate of their progeny over time, we crossed mice with an epidermal-specific photoactivatable H2B-mCherry41 transgene (K14-H2B-PAmCherry) to Pik3ca 2× mice. We then broadly activated oncogene expression using high-titre transduction with LV-Cre co-expressing membrane-associated green fluorescent protein (GFP) (LV-Cre-mGFP; Fig. 3a; Supplementary Fig. 5b). At P21, we photolabelled individual GFP-positive progenitors (Fig. 3b,c) and followed them as they divided (division 1). We tracked their daughter cells, which we scored as progenitors if they maintained mitotic potential, as evidenced by cell cycle re-entry (division 2), or as differentiated progeny if they left the basal layer and assumed flattened morphology (Fig. 3b,c). We observed that epidermal progenitors in wild-type skin undergo all three types of cell division, with asymmetric being more common (~50%) followed by similar rates of symmetric renewal (~25%) and differentiation (~25%, Fig. 3d). In contrast, symmetric renewal was significantly suppressed in oncogenic Pik3ca-expressing epidermis, whereas asymmetric division and symmetric differentiation were increased (Fig. 3d).
To investigate whether oncogene-driven differentiation is a cell autonomous process, we transduced Cre-reporter mice with LV-CreER and treated them with tamoxifen at P19 (Fig. 3e). We confirmed that a low dose of the drug resulted in sporadic labelling of epidermal progenitors and efficient activation of oncogenic Pik3ca expression (Supplementary Fig. 5c–h). At 48 h, we observed that most labelled progenitors had divided, and we scored their daughter cell fates (Fig. 3f). As with ubiquitous oncogene activation, symmetric renewal was significantly reduced, and both symmetric differentiation and asymmetric divisions significantly increased in Pik3ca 2× cells compared with wild-type (Fig. 3g).
Using three independent differentiation assays, we established that the progenitor renewal rate in Pik3ca 2× epidermis is significantly lower than in the wild-type epidermis (0.52 versus 0.43; Supplementary Fig. 5i), and we expected this difference to have a profound effect on tissue growth12. Indeed, clonal expansion in epidermis under constant proliferation and tissue turnover rate (based on a long-term EdU chase assay and on previous results41) is predicted to vary between homeostasis and rapid expansion or loss when renewal rates change by as little as 5–10% (Supplementary Fig. 6a,b). To test whether oncogenic Pik3ca-driven differentiation is sufficient to promote the loss of clonal expansion, we generated a model of clonal growth using proliferation and renewal rates observed in wild-type and Pik3ca 2× epidermis (Supplementary Fig. 6c). Our model predicted that single cell activation of oncogenic Pik3ca in the adult epidermis would result in clone loss, which was consistent with our long-term lineage tracing analysis (Fig. 4a,b). To test whether oncogene-induced differentiation can affect tumour initiation, we transduced wild-type and Pik3ca 2× epidermis with lentivirus expressing the human papillomavirus (HPV) E7 oncogene. We found that progenitor renewal and clonal expansion were significantly increased in HPV E7 transduced wild-type epidermis, and that tumour growth was initiated as early as 1 month (Fig. 4c–f). Importantly, oncogenic Pik3ca significantly suppressed progenitor renewal and clonal expansion and delayed tumour initiation driven by HPV E7 (Fig. 4c–f). This result suggests that constitutively activated PI3K signalling can act as a tumour suppressor during epidermal tumour initiation.
The AKT substrate SH3RF1 mediates PI3K-driven differentiation
To functionally dissect the molecular mechanism of oncogenic Pik3ca-driven differentiation, we focused on AKT as the dominant effector kinase of PI3K signalling, which is activated in Pik3ca 2× epidermis (Supplementary Fig. 2b). We selected shRNAs that efficiently deplete Akt1, Akt2 or Akt3 transcripts, and showed that silencing of Akt1 and Akt2, but not Akt3, significantly promoted progenitor renewal in both wild-type and Pik3ca 2× tissues (Supplementary Fig. 7a–c). Akt1 and Akt2 were similarly required for oncogenic Pik3ca-driven proliferation (Supplementary Fig. 7d,e). To test whether progenitor differentiation is regulated by a specific AKT substrate and independently of proliferation, we pooled 1,062 shRNAs targeting 242 validated AKT substrates42, and performed two genetic screens in vivo (Fig. 5a). For the differentiation screen, we collected basal (α6 Itghigh) and suprabasal (α6 Itglow) epidermal cells (Fig. 5a). For the proliferation screen, we isolated dividing (EdU-positive) and non-dividing (EdU-negative) basal cells following an 8-h EdU pulse (Fig. 5a). We expected the following results: (1) shRNAs that inhibit differentiation drivers will be enriched in basal cells (pro-renewal; Fig. 5a,b); (2) shRNAs against drivers of progenitor renewal will be enriched in suprabasal cells (pro-differentiation; Fig. 5a,b); and (3) shRNAs that are dispensable for proliferation will be equally present in EdU-positive and EdU-negative progenitors (proliferation neutral; Fig. 5a,c).
We quantified the shRNA representation in each group of cells36 and identified Sh3rf1 as the top candidate whose silencing promoted differentiation without an effect on proliferation (Fig. 5b–d; Supplementary Tables 2, 3). To test whether SH3RF1 is an AKT substrate in the epidermis, we pulled down endogenous protein and probed it using an antibody against the phospho-AKT substrate. We detected AKT phosphorylation of SH3RF1 in wild-type epidermis that was further increased in Pik3ca 2× tissue (Fig. 5e). We observed the same result when full-length SH3RF1 (SH3RF1WT) was expressed from a lentivirus (Fig. 5f); these results confirm that AKT phosphorylates SH3RF1 in vivo. To functionally test SH3RF1 as a specific differentiation regulator, we performed a series of gain- and loss-of function experiments in vivo. To model the AKT phosphorylation-mediated suppression of SH3RF1, we transduced wild-type epidermis with shRNA constructs that most efficiently depleted its transcript (Supplementary Fig. 7f). Sh3rf1 depletion promoted progenitor differentiation without a significant effect on cell division, thus validating our genetic screens (Fig. 5g). To counter the effect of AKT phosphorylation, we overexpressed SH3RF1WT in Pik3ca 2× epidermis (Fig. 5h). To control for a possible reduction in AKT phosphorylation that may be caused by substrate overexpression, we also expressed a known AKT substrate, H3F3B. We observed that SH3RF1 expression, but not H3F3B, suppressed oncogenic Pik3ca-driven differentiation while proliferation was unchanged (Fig. 5h). Finally, to test whether SH3RF1 can regulate oncogene-driven clonal expansion, we overexpressed SH3RF1WT in wild-type and Pik3ca 2× epidermis and observed that the resultant clones were significantly larger and were maintained long-term compared with Pik3ca 2× epidermis alone (Fig. 5i,j). Together, our data suggest that SH3RF1 is an AKT substrate in the epidermis, where it acts as a specific mediator of oncogenic Pik3ca-driven differentiation and clonal growth.
Activated PI3K–AKT inhibits SH3RF1-mediated support of JNK signalling to promote progenitor differentiation
SH3RF1 is a SH3 domain-containing scaffold molecule necessary for JNK signalling43. SH3RF1 can also function as an E3 ubiquitin ligase to regulate additional pathways44,45. Importantly, the scaffold function of SH3RF1 is independent of its E3 ligase activity, and SH3RF1 with a deleted RING finger domain (SH3RF1ΔRing) can still support JNK signalling46,47. In addition, AKT-mediated phosphorylation of SH3RF1 only disrupts its scaffold function and not its ability to activate JNKs43,46,47,48,49. Therefore, we hypothesized that SH3RF1 function in oncogene-driven differentiation depends on its scaffold function in the regulation of JNK signalling. We generated an allelic series of SH3RF1 constructs (Fig. 6a,b) and expressed them in wild-type and Pik3ca 2× epidermis. We found that expression of SH3RF1ΔRing was equal to that of SH3RF1WT in promoting progenitor renewal and clonal expansion independently of proliferation (Fig. 6c–e, Supplementary Fig. 7g), confirming that the E3 ubiquitin ligase function of SH3RF1 is dispensable in oncogene-induced differentiation.
We next expressed AKT phosphorylation site (S304) mutated SH3RF1SA as well as phosphomimetic SH3RF1SD in wild-type and Pik3ca 2× epidermis (Fig. 6a,b). We observed that SH3RF1SD had no effect on progenitor renewal or clonal growth, while SH3RF1SA significantly promoted both processes (Fig. 6c–e; Supplementary Fig. 7g). These observations suggest that clonal expansion and the progenitor renewal-promoting ability of SH3RF1 is dependent on, and suppressed by, AKT-mediated phosphorylation.
SH3RF1 promotes JNK signalling as a scaffold for assembly of the MLK–MKK complex46,49. This process has been demonstrated to be negatively regulated by AKT-mediated phosphorylation on S304 of SH3RF146,49. Meanwhile, JNK has been implicated as a positive regulator of progenitor renewal in skin50,51. Together, they suggest a molecular pathway whereby AKT phosphorylation and suppression of SH3RF1 inhibits the JNK signalling-mediated maintenance of progenitor cell fate (Fig. 7a). Consistent with our model, a gene set enrichment analysis (GSEA) of the oncogenic Pik3ca transcriptome demonstrated a significant suppression of JNK signature genes compared with the wild-type transcriptome (Fig. 7b). Moreover, immunofluorescence staining of epidermal tissues showed that phospho-JNK was significantly reduced in basal progenitors expressing oncogenic Pik3ca, and that the staining is largely recovered upon SH3RF1WT expression (Fig. 7c; Supplementary Fig. 7h). To directly test whether PI3K–AKT-mediated phosphorylation of SH3RF1 can modify epidermal JNK signalling, we performed a series of experiments. Western blot analyses showed that JNK phosphorylation is suppressed in Pik3ca 2× epidermis relative to wild-type, and that this suppression can be rescued by the overexpression of SH3RF1WT (Fig. 7d). SH3RF1ΔRing also rescued JNK phosphorylation, and expression of SH3RF1SA but not SH3RF1SD rescued and further enhanced phospho-JNK level in Pik3ca 2× epidermis. These results suggest that SH3RF1 promotes JNK signalling independently of its E3 ubiquitin ligase function, and that this positive regulation can be suppressed by AKT phosphorylation (Fig. 7e).
Last, we tested whether suppression of JNK signalling is necessary and/or sufficient for oncogenic Pik3ca-induced differentiation. We overexpressed the JNK activator43,48,52 MLK1 (also known as MAP3K9) in Pik3ca 2× epidermis and observed increased phospho-JNK levels (Fig. 7f) and suppression of oncogenic Pik3ca-induced differentiation with no effect on proliferation (Fig. 7g; Supplementary Fig. 7i). This supports a model whereby JNK signalling is downstream of oncogenic Pik3ca in regulating epidermal progenitor renewal, and that its activation is sufficient to overcome oncogene-induced differentiation. To test whether the suppression of JNK is necessary for oncogene-induced differentiation, we expressed SH3RF1SA in Pik3ca 2× epidermis, while at the same time we depleted Jnk1 using shRNA knockdown (Fig. 7h). We observed that silencing of Jnk1 significantly abrogated the rescue effect of SH3RF1SA on oncogenic Pik3ca-induced differentiation without affecting cell division (Fig. 7i; Supplementary Fig. 7j), confirming that JNK signalling is not only required for but is the dominant mediator of SH3RF1 function in regulating differentiation. Together, we demonstrate that the AKT-mediated phosphorylation of SH3RF1 and the subsequent suppression of JNK signalling is a critical pathway in oncogenic Pik3ca-induced differentiation, and provide a mechanism for oncogenic tolerance without triggering cell cycle arrest or cell death in the skin epithelium.
How tissues respond to oncogenic mutations to prevent clonal expansion has been a central question in cancer research for decades8,53,54. Senescence and apoptosis can block oncogene-driven clonal expansion in culture7,55, and senescence has been demonstrated to have a robust tumour-suppressive potential under certain physiological scenarios4,5,56. However, both of these cellular processes feature an abrupt block to proliferation and tissue disruption, which is inconsistent with evidence that skin epithelium, despite a large quantity of oncogenic lesions3, can maintain both its proliferative and functional integrity. Thus, it is likely that skin, which depends on high rates of tissue turnover to maintain an efficient barrier to the external environment, employs a tumour-suppressive mechanism that can maintain proliferation and manage long-term expansion of oncogene-activated cells. The oncogenic Pik3ca-induced differentiation that we describe here fits these requirements, as it allows the epidermis to manage oncogenic stress by driving excess oncogene-activated cells out of the mitotic progenitor pool.
Even though lineage choice in epidermis is relatively simple, and differentiation is accompanied by observable and well-documented changes in cell morphology and marker expression, methods to directly assess cell fate choice in vivo have been slow to emerge. Indeed, investigations of how oncogenes affect cell fate choices and our discovery of oncogenic Pik3ca-induced differentiation were made possible only recently through the development of direct and quantitative assays, including intravital imaging41. In the current study, we add to this experimental toolbox by describing a simple EdU–BrdU pulse–chase assay as a sensitive method to quantify the progenitor cell renewal rate in skin epithelium.
Animal studies have already shown that activation of the PI3K–AKT pathway in mice through oncogenic mutations in Pik3ca31,32 or loss of Pten57,58 does not result in epidermal tumorigenesis59, even though tumours are initiated in multiple other epithelial organs. These observations suggest that the ability of oncogenic Pik3ca to promote tumorigenesis may be stringently restricted by a tumour-suppressive mechanism in distinct epithelia. Curiously, differentiation that is accompanied by a loss of proliferative potential is not seen across all epithelia, but seems specific for highly proliferative ones, such as skin epidermis60,61, intestinal epithelium62 and oral mucosa11. Future studies should explore whether these tissues employ differentiation to block clonal expansion and to counter oncogenic stress.
Our identification of differentiation as the dominant growth-suppressive mechanism in skin epithelium further suggests that its loss in the context of oncogenic lesions would provide oncogene-activated clones with the ability to expand while retaining higher proliferation rates. Supporting this notion, mutations identified as prominent clonal expansion promoters in both human tissue3 and our genetic screen are always gained earlier in SCC progression than in oncogenic lesions for PIK3CA63.
We used equal numbers of male and female animals throughout the study. Pik3caH1047R/H1047R mice (donated by W. A. Phillips31,32,33), K14-H2B-PAmCherry mice (donated by V. Greco41), and R26mT/mG and R26yfp/yfp Cre-reporter mice (Jackson Laboratories) were on the C57BL/6 or C57BL/6-Tyrc-2J/J background. Mice were housed and cared for in an AAALAC-accredited facility at the Fred Hutchinson Cancer Research Center, and all animal experiments were conducted in accordance with ethical regulations of the Fred Hutchinson Cancer Research Center and IACUC-approved protocols (project licence number 50814).
Lentivirus production and transduction
Large-scale production and concentration of lentivirus were performed as previously described26. Detailed descriptions of the lentiviral transduction of 293T cells (Invitrogen) and primary keratinocytes in culture, and of in utero-guided lentiviral transduction in vivo can be found elsewhere26,27,64.
ORF overexpression was achieved using a pLX EF1 Barcode vector (Supplementary Fig. 1b). The pLX EF1 Barcode vector was modified from pLX302 (Addgene), in which the following were replaced: (1) the PGK-puroR cassette between PpuM1/Xba1 sites with a randomized 10-bp barcode; (2) the CMV promoter between Xho1/Nde1 sites with an EF1α promoter from pEF-BOS (Addgene). RNA interference-mediated gene depletion was achieved using pLKO1 shRNA vectors from the mouse TRC1.0 shRNA library (Sigma-Aldrich). To construct the lentiviral pools and to ensure equal lentivirus representation, plasmids were mixed together in equal molar ratios. Expression of Cre-recombinase in tandem with ORF expression was achieved using pLX EF1 Cre vectors. This vector was modified from pLX302 (Addgene) by inserting Cre downstream of the PGK promoter using Kpn1/Xba1 sites. Cre shRNA expression was achieved using pLKO-Cre vectors as previously described26,27.
In vivo genetic screens
To quantify construct abundance in the screen for clonal expansion, head skin of mice at P21 was collected and digested in 0.25% collagenase at 37 C° for 1 h to release mesenchymal cells from the epithelium. Head skins from two animals were pooled and treated as a biological replicate to achieve an ~200-fold coverage. To quantify construct abundance in the renewal and cell division screens, head skin of mice at embryonic day 18.5 (E18.5) was digested in 2 mg ml–1 dispase at 37 C° for 1 h to separate epidermis from dermis. Epidermal tissue was further digested with 0.25% trypsin for 30 min into single cells. Head skins from four animals were pooled together to make one biological replicate, thus achieving an ~45-fold coverage. For the renewal screen, single epidermal progenitor cells were stained with CD326/EpCAM-APC (G8.8, 1:50; BD Biosciences) and CD49f/α6-integrin-PerCP (GoH3, 1:50; BioLegend). For the cell division screen, single epidermal cells were subjected to Click-iT EdU detection (C10338, Invitrogen) followed by CD49f/α6-integrin-PerCP staining. Cell populations of interest were isolated using a BD FACSAria II machine (BD Biosciences). Genomic DNA from all samples was extracted using a QIAamp DNA tissue mini kit (Qiagen). Barcode pre-amplification, sequencing and data processing using the Deseq2 program65 were performed as previously described27,66.
Intravital imaging using two-photon microscopy
Intravital imaging was performed using a LSM 780 multiphoton laser scanning confocal microscope (Zeiss) according to a previously described protocol41. The animal was immobilized using a modified custom-made device67 to minimize vibration from breathing during imaging of the head skin. To photoactivate PA-H2B-mCherry, we exposed the cells for 2 min in a continuous scan with a 810-nm laser set at 3%. Membrane-associated GFP and Tomato signals were captured using a 940-nm laser, while the PA-H2B-mCherry signal was captured using a 1,040-nm laser.
E18.5 epidermis was snap-frozen using liquid nitrogen, mechanically pulverized and lysed in lysis buffer (150 mM NaCl, 10 mM HEPES buffer, pH 7.4, and 1% Nonidet P-40). Lysates were incubated with anti-V5 affinity beads (A7345, Sigma-Aldrich) or dynabeads (10003D, Invitrogen) pre-incubated with anti-SH3RF1 antibody (3H3, Abnova) overnight at 4 °C. Beads with affinity-bound proteins were washed six times with immunoprecipitation wash buffer (200 mM NaCl, 10 mM HEPES, pH 7.4, and 0.1% Nonidet P-40) before direct loading onto a 4–12% gel and subjected to western blot analyses.
Immunofluorescence and western blot analyses
The following primary antibodies were used: chicken anti-GFP (1:1,000 for immunofluorescence; ab13970, Abcam); mouse anti-BrdU (1:100 for immunofluorescence; MoBU-1, Invitrogen); mouse anti-β-actin (1:3,000 for western blotting; 66009, Proteintech); mouse anti-V5 (1:3,000 for western blotting; V5-10, Sigma-Aldrich); rabbit anti-K10 (1:1,000 for immunofluorescence; Poly19054, BioLegend); rabbit anti-phospho-AKT (1:1,000 for western blotting; D9E, Cell Signaling); rabbit anti-total AKT (1:500 for western blotting; C67E7, Cell Signaling); mouse anti-SH3RF1 antibody (1:400 for western blotting; 3H3, Abnova); rabbit anti-phospho-AKT substrate (1:1,000 for western blotting; 110B7E, Cell Signaling); rabbit anti-phospho-SAPK/JNK (1:500 for western blotting, 1:100 for immunofluorescence; 81E11, Cell Signaling); and rabbit anti-total SAPK/JNK (1:1,000 for western blotting; 9252, Cell Signaling). Tissues were processed for immunostaining as previously described26,27 and mounted in ProLong Gold with or without 4,6-diamidino-2-phenylindole (DAPI; Life Technologies). For EdU–BrdU pulse–chase differentiation assays, tissue sections were first processed for EdU Click-iT following the manufacturer’s instructions. Next, tissues were fully processed for K10 and GFP immunofluorescence detection. Last, tissue sections were incubated in 2 N HCl at 37 °C for 30 min to denature DNA, quenched with 0.1 M sodium borate, pH 8.5, twice, and processed for BrdU immunofluorescence detection. Confocal images were taken on a Zeiss LSM700 system using a Plan-Apochromat ×40/1.4 oil objective. Images were processed using Zeiss Zen and ImageJ software. Western blotting was performed using a Novex system (Invitrogen), and chemiluminescent signals were captured using an Odyssey Fc system (LI-COR).
EdU–BrdU cell division assay and quantification
To measure the average cell cycle interval in epidermis, we developed a cell cycle assay based on the specific labelling of cells that have completed cell division within a defined time frame. By using such a method, we minimized the potential artefact of conventional nucleotide incorporation-based proliferation assays; that is, tissues with a longer S phase but not an essentially faster division rate can appear to have more nucleotide incorporation (Supplementary Fig. 2d). To ensure the specificity of the assay, we tested the BrdU-specific antibody MoBU-1 and confirmed that animals injected with BrdU were readily labelled, while EdU (or sham)-injected animals showed no staining. Similarly, we detected EdU using Click-iT chemistry only in animals injected with EdU.
To perform the assay, as illustrated in Supplementary Fig. 2d, we first administered EdU to animals followed by BrdU injection 2 h later. We expected that cells that have completed the S phase during the first 2 h would incorporate EdU only, while cells subsequently going through S phase would have incorporated both EdU and BrdU (Supplementary Fig. 2d). Six hours later, we collected epidermis and detected EdU and BrdU signals by immunofluorescence. At this time point, we expected cells that have completed the S phase during the initial 2 h and subsequently divided would give rise to two EdU-positive-only progenies, while cells that stalled in G2 would result in one EdU-positive-only cell (Supplementary Fig. 2d). However, we found that over our time frame, most cells divide (~97%) and that only a minority in both wild-type and Pik3ca 2× epidermis remains in G2 phase (Supplementary Fig. 3c,d).
To calculate the cell division interval, we defined the number of EdU-positive-only cells as E. As one cell division gives rise to two daughter cells, the number of cell divisions that give rise to EdU-positive-only cells is E/2. As we have demonstrated, only basal cells have the potential to divide in the epidermis (Supplementary Fig. 3a,b); therefore, the fraction (F) of basal cells that divide within 2 h to give rise to EdU-positive-only cells is F = (E/2)/B, where B is the total number of basal cells. We extrapolated this measure to the entire basal cell population by using the following ratio: D/1 = 2 h/F, where D is the average cell division interval (in hours).
EdU–BrdU pulse–chase differentiation assay and quantification
Since we can uniquely label a population of cells (EdU-positive only) that had undergone S phase within the initial 2 h and subsequently divided as described above, we next set out to determine how long after cell division an average daughter cell takes before starting to express the differentiation marker K10. We administered EdU and then BrdU and collected tissues every 2 h. Tissues were processed to detect EdU and BrdU together with K10 (to mark committed progeny). Our analysis showed that K10 expression was detected as early as 2 h and plateaued by 6 h of BrdU labelling. Importantly, similar dynamics were observed in both wild-type and Pik3ca 2× tissues and irrespective of which nucleoside analogue was administered first. Thus, by analysing epidermis 6 h after the start of BrdU labelling, we can calculate the rate of progenitor cell renewal of the EdU-positive-only cells using the following equation: rate of renewal = number of EdU-positive-only K10-negative cells/total number of EdU-positive-only cells.
RNA-seq and GSEA analyses
We isolated inter-follicular epidermis from dermis and hair follicles using dispase digestion. Single cells were obtained following 15 min of trypsin digestion and then stained for basal integrin expression64. RNA from FACS-isolated α6 Itghigh epidermal progenitors was extracted using TRIzol LS (Invitrogen) and a phenol–chloroform protocol. The extracted RNA was then purified using a QIAamp RNA mini kit (Qiagen) as per the manufacturer’s instructions. RNA quality was assessed using an Agilent 2100 Bioanalyzer, with all samples passing the quality threshold of RNA integrity number (RIN) score of >8. The library was prepared using an Illumina TrueSeq mRNA sample preparation kit at the Fred Hutchinson Cancer Research Center Genomic Core Facility, and complementary DNA was sequenced on an Illumina HiSeq 2000. Reads were mapped to mm9 build of the mouse genome using TopHat, and transcript assembly and differential expression were determined using Cufflinks68. GSEA69 was performed using GSEA2. Reference gene expression signatures were obtained from the following previously published expression profiles: HFSC34; mouse basal/suprabasal cells35; human basal/suprabasal cells36; JNK activation signature70.
Statistics and reproducibility
All quantitative data are expressed as the mean ± s.d. Differences between groups were assayed by two-tailed Student’s t-test or one-way analysis of variance using Prism 5 (GraphPad software). Differences were considered significant when P < 0.05. All quantitative data were collected from experiments performed in at least three samples or biological replicates. The sample size was not predetermined and the experiments were not randomized.
Further information on experimental design is available in the Nature Research Reporting Summary linked to this article.
RNA–seq data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under accession code GSE99659. Previously published microarray data that were re-analysed here are available under accession codes GSE4170434, GSE4885935, GSE2605936 and GSE5053070. Source data for Figs. 1e, 2b–d, f, 3d, g, 4a, c, e, 5g, h, j, 6c, e, 7g, i and Supplementary Figs. 2c, 3a, d, 4a–c, e, 5c–e, i, 7a–g, i, j are provided in Supplementary Table 4. All other data supporting the findings of this study are available from the corresponding author upon reasonable request.
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The authors thank the following: W. Phillips for sharing the inducible Pik3caH1047R mouse model; V. Greco and C. Pineda for sharing the photoactivatable mouse model and the intravital imaging technique; M. Schober and A. Hsieh for critical reading of the manuscript; the Comparative Medicine Department (AAALAC-accredited; R. Uthamanthil, Director) for care of the mice in accordance with National Institutes of Health (NIH) guidelines; the Genomics Department (J. Delrow, Director) for sequencing; the Scientific Imaging Department (J. Vazquez, Director) for advice; and the Flow Cytometry Department (A. Berger, Director) for flow cytometry and FACS. This research was supported by grants from the NIH (R01-AR070780 to S.B.), a Cell & Molecular Biology Training Grant (to M.S.), and a Thomsen Family Fellowship (to Z.Y.). The authors dedicate this work to X. Chen, and are grateful for her strength and support.
The authors declare no competing interests.
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Integrated supplementary information
(a), Catalog of 33 most common cancer-driver genes with oncogenic lesions (copy number alteration and mutation) across squamous cell carcinomas (SCC). (b), Lentiviral constructs for expression of ORFs and shRNAs. Barcode in the pLX EF1 vector and shRNA in the pLKO1 vector are used for sequencing-based identification and quantification of individual gene-targeting constructs. (c), Correlation between protein expression of the V5-tagged ORF library (V5/Actin) in 293 T cells and primary mouse keratinocytes (KRT). Control ORF (GFP) is in blue. (d), Ten most commonly mutated oncogenes in epithelial cancer. Statistics are based on TCGA data for carcinomas of the bladder, bowel, breast, head & neck, kidney, lung, ovary, pancreas, cervix, esophagus, stomach, liver, prostate, thymus, thyroid and uterus tissues. SCCs included SCC of the head & neck, lung and cervix. Each cancer type was equally weighted in the statistics.
(a), Epidermal transduction with LV-Cre results in efficient activation of Pik3caH1047R expression. (b), Western blot demonstrates increased phosphorylation of AKT in LV-Cre transduced skin epithelium expressing PIK3CAH1047. Experiment was repeated 3 times independently with similar results. Unprocessed blots see Supplementary Fig. 8. (c), Epidermal expression of PIK3CAH1047R results in faster cell division at all stages of tissue growth and homeostasis. Statistics based on n = 3 animals of each condition. Error bar: SD, center value: mean. (d), Schematic comparison of how theoretical differences in the length of individual cell cycle stages may affect EdU-BrdU pulse chase labeling, and how to calculate cell division interval from the observed numbers. Assay specifically labels cells which complete S-phase during the initial 2hrs of EdU pulse. The length of the G1/S phase length does not, while significant arrest in G2 may affect the number of EdU+ only cells at the assay time point. Statistical source data for (c) are shown in Supplementary Table 4.
Supplementary Figure 3 EdU-BrdU pulse chase differentiation assay measures renewal rate in epidermis.
(a) and (b), Statistics (a) and representative images (b) show that short (2 hour) pulse of EdU results in cell labeling that is restricted to the K10- progenitor cell population in wild type (WT) and oncogenic Pik3ca-expressing epidermis. Statistics based on n = 3 animals of each condition. Error bar: SD, center value: mean. Scale bar, 50 μm. (c) and (d), Representative flow plot (c) and statistics (d) show that in the EdU+ only population isolated from EdU-BrdU pulse-chase labeled epidermis, only ~3% cells are arrested at G2 (2n population), and there is no significant difference in the size of the G2 arrested population in WT and Pik3ca 2X epidermis. Statistics based on n = 3 WT animals and 5 Pik3ca 2X animals. Two-tailed t test, P-value as indicated. Error bar: SD, center value: mean. (e), Representative cell labeling in the EdU-BrdU pulse-chase assay. Experiment was repeated 3 times independently with similar results. EdU marks cells that have replicated their DNA over the 8 hours of the experiment. BrdU labels replicating cells during the last 6 hours of it. EdU+ only expression marks daughter cells that were given sufficient time to commit to progenitor or differentiated fate, as reported by expression of differentiation marker K10. Scale bar, 50 μm. Statistical source data for (a,d) are shown in Supplementary Table 4.
(a-c), Clonal and ubiquitous (field) activated Pik3caH1047R expression in skin does not affect epithelial cell crowding (a), and results in equal rates of renewal (b) and cell division (c). However, the rate of renewal is elevated in WT cells adjacent to Pik3ca 2X clone ( ≤ 5 cells from Pik3ca 2X clone) but not in WT cells that are non-adjacent to Pik3ca 2X clones. Statistics based on n = 3 animals of each condition. Two-tailed t test, P-value as indicated. Error bar: SD, center value: mean. (d), Staining of differentiation marker K10, loricrin and filaggrin exhibited a similar pattern in WT and Pik3ca 2X tissue. Experiment was repeated 3 times independently with similar results. Scale bar, 50 μm. e, Long term EdU chase assay started at P21 shows that tissue turnover rates are similar between WT and Pik3ca 2X epidermis. Statistics based on n = 3 animals of each condition. Two-tailed t test, P-value as indicated. Error bar: SD, center value: mean. (f), GSEA shows that oncogenic Pik3ca biases gene expression in epidermal progenitors towards differentiation. Statistics are based on n = 3 animals of each condition. ES, NES, FDR generated by GSEA2 program. Statistical source data for (a-c,e) are shown in Supplementary Table 4.
Supplementary Figure 5 Oncogenic Pik3ca promote differentiation via suppression of progenitor self-renewal.
(a), Set-up for live imaging of head skin epidermis. (b), Visualization of LV-Cre-mGFP transduced cells in vivo. Arrows indicate hair follicles. Suprabasal cells are identified based on their flattened morphology. Basal cells are identified based on their cuboidal morphology and proximity to the basement membrane (blue; second harmonic generation signal from Collagen). Scale bar, 50 μm. Experiment was repeated in transduced head skin of 4 animals independently with similar results. (c-e), Activation rate and bias of LV-CreER transduced epidermis treated with low dosage of Tamoxifen. At P20, CreER activity, based on conversion of mT to mG, is detected in ~1% of epidermal cells (c), majority of clones (~90%) are found as single cells (d), and most of the single cells ( > 98%) are localized to the basal/progenitor layer (e). Statistics based on n = 7 WT animals and 6 Pik3ca 2X animals. Error bar: SD, center value: mean. (f), FACS plot of GFP+/- cells sorted from Pik3ca 2X R26mT/mG epidermis transduced with LV-Cre or with LV-CreER and treated with low dose of Tamoxifen. Experiment was repeated 3 times independently with similar results. (g), Schematic of genotyping primers31 detecting substitution of wild type exon 20 with H1047R exon 20 of Pik3ca. (h), Genotyping PCR shows that low dose of Tamoxifen can efficiently activate Pik3caH1047R. Experiment was repeated 3 times independently with similar results. (i), Rate of progenitor cell renewal in WT and Pik3ca 2X epidermis observed using direct two-photon imaging is consistent with EdU-BrdU pulse-chase assay quantification. Statistics based on n = 3 animals of each condition. One-way ANOVA, P-value as indicated. Error bar: SD, center value: mean.
Supplementary Figure 6 Suppressed progenitor renewal rate predicts long-term loss of oncogenic Pik3ca-expressing clones.
(a), Schematic of clonal growth in epidermis started from a single cell with rate of renewal = 0.5, division interval = 1 day, and tissue turnover rate = 10 days. (b), Theoretical growth pattern of epidermal clones started from a single basal cell, and with a constant division interval and tissue turnover rate = 10 days (based on long term EdU chase and previous publication41). Rate of progenitor renewal is in the physiological range of 0.4-0.6. (c), Theoretical growth pattern of wild type and Pik3ca 2X clones started as a single cell at P20. Rates of differentiation, proliferation and tissue turnover are as observed in 3 independent differentiation and tissue turnover assays.
(a), mRNA expression of Akt1-3 in keratinocytes transduced with control shScram or gene-targeting shRNAs. Two shRNAs for each gene that induced strongest transcript depletion (marked with red arrow) were used for functional studies in vivo. Experiment was repeated 3 times independently with similar results. Error bar: SD, center value: mean. (b-e), Depletion of Akt1-3 shows that Akt1 and Akt2 are required for epidermal differentiation and cell division phenotypes. EdU-BrdU pulse-chase assay shows that Akt1 and Akt2 are required for differentiation in Pik3ca 2 × (b) and WT (c) skin epithelium. Cell division rate is equally dependent on Akt1 and Akt2 in Pik3ca 2 × (d) and WT (e) epidermis. Statistics based on n = 3 animals of each condition. Two-tailed t test, P-value as indicated. Error bar: SD, center value: mean. (f), Sh3rf1 mRNA expression in keratinocytes transduced with gene-targeting shRNAs relative to control shScram. Two most efficient shRNAs (red arrows) were used in subsequent gene-depletion studies. Experiment was repeated 3 times independently with similar results. Error bar: SD, center value: mean. (g), SH3RF1 mutants do not affect cell division rate in both wild type and Pik3ca 2X epidermis. Statistics based on n = 3 animals of each condition. Two-tailed t test, P-value as indicated. Error bar: SD, center value: mean. (h), Immunofluorescence staining of phosphorylated JNK (red) in clonally activated Pik3ca 2X epidermis (green). Transduction with LV-Cre reduces p-JNK levels (top rows) that are rescued by overexpression of Sh3rf1 (bottom rows). Inset boundaries are marked by white rectangle in larger view images. Dotted line marks epidermal/dermal boundary. Dashed line marks the interface between Cre-transduced cells. Scale bar, 25 μm. Experiment was repeated 3 times independently with similar results. (i), MLK1 does not affect cell division rate in Pik3ca 2X epidermis. (j), Knockdown of Jnk1 does not affect cell division rate in Pik3ca 2X epidermis expressing SH3RF1SA. Statistics based on n = 3 animals of each condition. Two-tailed t test, P-value as indicated. Error bar: SD, center value: mean.
Uncropped Western blots presented in the current study.
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Ying, Z., Sandoval, M. & Beronja, S. Oncogenic activation of PI3K induces progenitor cell differentiation to suppress epidermal growth. Nat Cell Biol 20, 1256–1266 (2018). https://doi.org/10.1038/s41556-018-0218-9
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