EGFR signalling as a negative regulator of Notch1 gene transcription and function in proliferating keratinocytes and cancer - PubMed (original) (raw)

EGFR signalling as a negative regulator of Notch1 gene transcription and function in proliferating keratinocytes and cancer

Vihren Kolev et al. Nat Cell Biol. 2008 Aug.

Erratum in

Abstract

The Notch1 gene has an important role in mammalian cell-fate decision and tumorigenesis. Upstream control mechanisms for transcription of this gene are still poorly understood. In a chemical genetics screen for small molecule activators of Notch signalling, we identified epidermal growth factor receptor (EGFR) as a key negative regulator of Notch1 gene expression in primary human keratinocytes, intact epidermis and skin squamous cell carcinomas (SCCs). The underlying mechanism for negative control of the Notch1 gene in human cells, as well as in a mouse model of EGFR-dependent skin carcinogenesis, involves transcriptional suppression of p53 by the EGFR effector c-Jun. Suppression of Notch signalling in cancer cells counteracts the differentiation-inducing effects of EGFR inhibitors while, at the same time, synergizing with these compounds in induction of apoptosis. Thus, our data reveal a key role of EGFR signalling in the negative regulation of Notch1 gene transcription, of potential relevance for combinatory approaches for cancer therapy.

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Figures

Fig. 1

Fig. 1. Negative control of Notch1 activity and expression by EGFR/ERK/AP1 signaling

A and B : Primary human keratinocytes (HKCs) were treated with AG1478 at the indicated concentrations or with recombinant EGF (1.0 ng/ml) for 24 hours. Hes1, Hes5 and Herp1 (CHANGE NAME IN THE FIG. FROM HEY2 TO HERP1) (A) and Notch1 (B) mRNA levels were quantified by real-time RT-PCR. Values are expressed as relative units after internal normalization for 36B4 mRNA levels, with similar results being obtained after normalization for β-actin mRNA. C : HKCs were treated with AG1478 and EGF as in the previous panel, followed by immunoblot analysis for Hes1, total and activated Notch1, Notch2, and γ-Tubulin as equal loading control. D : HKCs were transfected with two different siRNAs specific for EGFR in parallel with scrambled siRNA control. Expression of the EGFR, Hes1 and Notch1 genes was assessed, 48h after transfection, by real time RT-PCR analysis (left panels), and confirmed at the protein level by immunoblotting (right panels). E and F : HKCs were transfected with validated siRNAs for the indicated protein kinase (E) and transcription factor (F) genes, in parallel with scrambled siRNA control. >80% knock down efficiency was obtained for each of these genes, as verified by real time RT-PCR analysis with the corresponding specific primers 48h after transfection (data not shown). Levels of Notch1 mRNA expression were assessed by real time RT-PCR analysis as in the previous panels. For ERK1, ERK2, c-Jun, c-Fos and ELK1, similar results were obtained after transfection with a second set of specific siRNAs. G : HKCs, transfected with siRNA specific for c-Jun and scrambled siRNA control as in the previous panel, were analyzed by immunoblotting for total Notch1 and γ-Tubulin as equal loading control. Data were quantified by densitometric scanning, using the γ-Tubulin signal for normalization (right panel).

Fig. 2

Fig. 2. Suppression of EGFR signaling induces Notch1 expression through p53

A : HKCs were transfected with siRNAs specific for p53 plus/minus siRNAs for EGFR in parallel with scrambled siRNA control. Cells were analyzed 48 hours after transfection. “Knock down” of p53 expression was confirmed by real time RT-PCR and immunoblot analysis (left panels). Hes1 and Notch1 expression was assessed by real time RT-PCR as for the previous experiments. B : HKCs were infected with the lentiviral reporter vectors pTRH4-N1-2.4 (N1-2.4) and pTRH4-N1-0.4 (N1-0.4), carrying an internal luciferase gene driven by either a 2.4 or 0.4 kb region of the human Notch1 promoter (nucleotides −2472 to −1 and −392 to −1 from the initiation codon, respectively), which contains and lacks, respectively, the mapped p53 binding sites, . The pTRH4 vector, carrying the luciferase gene devoid of exogenous promoter, was used as control. HKCs stably infected with these viruses were transfected with siRNAs specific for p53 or scrambled siRNAs, followed by treatment (for 24 hours) with AG1478 (2 μM) or DMSO control, as indicated. For each set of cells, values are expressed as relative luciferase activity after protein normalization. C : HKCs were treated with AG1478 (2 μM) or DMSO control for 24 hours, followed by real time RT-PCR analysis of p21WAF1/Cip1 and Gadd45α expression. D : HKCs treated with AG1478 or DMSO as in the previous experiments, were analyzed by immunoblotting with antibodies against Mdm2, and γ-Tubulin as equal loading control. E : HKCs were treated with AG1478 and Nutlin (2 μM) for 24 hours, individually and in combination, followed by immunoblot analysis of p53 and γ-Tubulin expression. F : HKCs were treated with AG1478, transfected with EGFR-specific siRNA, or stimulated with EGF as in the previous experiments, and analyzed, in parallel with the corresponding controls, for levels of p53 mRNA expression by real time RT-PCR. G : HKCs were treated with AG1478 or DMSO control for 24 hours. Cells were then processed for chromatin immunoprecipitation analysis (ChIP) with an antibody against c-Jun and purified rabbit IgG as non-immune control. Real-time PCR of a distinct region of the human p53 gene promoter containing several conserved AP-1 binding sites (around position −2.6 kb, Site A in the map above) was performed along with PCR of a more downstream region devoid of such sites (around position −0.3 kb, Site B). The primers used to amplify these p53 promoter regions are listed in Supplemental Table II. H : cells were co-transfected with the p53n-Luc reporter plasmid, carrying a luciferase gene driven by a 2 kb region of the human p53 promoter, together with an expression vector for human c-Jun (c-Jun-pCMV,42) or empty vector control (left panel), or with siRNAs against c-Jun and scrambled siRNAs control. Luciferase activity was determined 48h later. For each set of cells, values are expressed as relative luciferase activity after protein normalization. I : HKCs were transfected with siRNAs against c-Jun in parallel with scrambled siRNA control (as in Fig. 1F, G) followed by measurement of p53 expression by real time RT-PCR. J : HKCs were transfected with siRNA against c-Jun either alone or in combination with siRNA against p53, in parallel with scrambled siRNA control. Notch1 expression was determined by real time RT-PCR as before.

Fig. 3

Fig. 3. Down-modulation of EGFR signaling induces keratinocyte differentiation through a Notch dependent mechanism

A : HKCs infected with a retrovirus expressing the dominant negative MAM51 peptide or GFP control (black and white bars, respectively) were treated with AG1478 (2 μM) or DMSO control for 24 hours. Expression of the Notch1, Hes1, Keratin 1 and Involucrin genes was assessed by real time RT-PCR. B : HKCs were treated with AG1478 (2 μM) and DAPT (10 μM) or DMSO control for 24 hours, followed by real time RT-PCR analysis of Hes1, Keratin 1 and Involucrin expression (left panel). Similarly treated cells were also analyzed for Keratin1 protein expression by immunoblot analysis, with γ-tubulin as equal loading control (right panels). C : HKCs were transfected with Notch1 or p53 specific siRNAs in parallel with scrambled siRNA control (black and white bars, respectively) and treated with AG1478 (2 μM) or DMSO followed by RT-PCR for Keratin1. “Knock down” of Notch1 expression was confirmed by real time RT-PCR and immunoblot analysis (left panels). D : HKCs were transduced with adenoviruses expressing activated form of Notch1 or GFP control and 24 h after infection stimulated with EGF (1 ng/ml). Expression of Keratin1 was assayed by RT-PCR analyses. E : HKCs were transduced with adenoviruses expressing Hes1 or GFP control followed by RT-PCR analysis of Keratin1.

Fig. 4

Fig. 4. Suppression of EGFR signaling induces p53 and Notch1 expression in intact mouse and human epidermis

A : Trangenic mice expressing a GFP reporter gene from a Notch/CBF1-responsive promoter were injected intraperitoneally with AG1478 (1 mg, in 100 μl 75% DMSO) or DMSO control every two days. 6 days later, mice were analyzed for GFP expression by immunofluorescence of back skin sections using anti-GFP antibodies. B and C : Back skin of the same mice as in the previous panel was isolated and the epidermis was separated from the underlying dermis by a brief heat treatment. Expression of the GFP transgene, as well as of the endogenous Notch1, p53 and keratin 1 genes was determined by real time RT-PCR, using β-Actin for internal normalization. D : Back skin epidermis of homozygous wa-2 mice (carrying a point mutation of the EGFR gene resulting in a > 90% fold decrease of EGFR activity, and heterozygous littermates was analyzed in parallel for endogenous p53 and Notch1 expression by RT-PCR as in the previous panel. E : skin biopsies from a cohort of melanoma cancer patients following treatment with the MEK inhibitor AZD6244 (ARRY-142886; ASTRA Zeneca) were analyzed, in parallel with biopsies from age- and gender-matched controls retrieved from the same body region, for expression of Notch1 expression by immunofluorescence analysis with anti-Notch1 antibodies. Same exposure and image capture conditions were utilized for all samples. In parallel with the increased Notch1 expression, immunostaining with anti-p53 antibodies demonstrated increased levels of this protein and data not shown). (F) : Freshly excised human skin samples were placed in semisolid medium and treated with DMSO control or AG1478 (10 μM) for 24h. Histological sections were analyzed by immunofluorescence with antibodies against the Notch1 protein. Arrows indicate the outermost layer of the epidermis and dotted line the innermost. H&E staining and immunofluorescence of parallel sections with anti-Keratin1 antibodies are shown in supplemental Fig. 5A. B : Freshly excised human skin samples, as in the previous panel, were placed in semisolid medium and treated with DMSO or AG1478 (10 μM) plus/minus DAPT (10 μM) (black and white bars, respectively) for 24h. The epidermis was separated from the underlying dermis by a brief heat treatment (60°C, 45”) followed by total RNA preparation and analysis of mRNA expression of the indicated genes by real time RT-PCR, using β-actin mRNA for normalization.

Fig. 5

Fig. 5. Differential Notch1 and p53 expression in the EGFR-dependent SOS-mouse skin tumor model plus/minus c-Jun deletion

A : Tumors formed by two K5-SOS-F transgenic mice with an intact c-Jun gene (Junf/f SOS+) and from two transgenics with a concomitant keratinocyte-specific deletion of the c-Jun gene (c-JunΔep SOS+) were analyzed by real time RT-PCR for levels of Notch1 and p53 expression (left and right panels, respectively), using β-actin for normalization. B : Extracts from another set of tumors, from three mice per genotype, was analyzed by immunoblotting with antibodies against the indicated proteins. C : Immunofluorescence/confocal analysis with anti-Notch1 antibodies of tumors formed by K5-SOS-F transgenics plus-minus c-Jun deletion. Hoechst staining was used for cell identification.

Fig. 6

Fig. 6. EGFR-dependent regulation of p53 and Notch in cancer cell lines and human squamous cell carcinomas (SCCs)

A – C: SCC12, SCC13 and SCCO28 cells, treated with AG1478 (2 μM) versus DMSO control for 24 hours, were analyzed for levels of p53 (A), p21WAF1/Cip1 (B), and Notch1 (C) expression by real time RT-PCR. D : SCCO28 cells were treated with AG1478 at the indicated concentrations for 24h followed by immunoblot analysis for Notch1 and γ-Tubulin as equal loading control. E : SCCO28 cells were transfected with p53-specific siRNA in parallel with scrambled siRNA control followed, 24 hours later, by treatment with AG1478 or DMSO for 24 hours. Real time RT-PCR was used to determine resulting levels of Notch1 expression, as well as to verify p53 knockdown (not shown). F : Surgically excised squamous cell carcinomas from five different patients (SCC1, 2, 3, 4, 5) were cut into small pieces of similar size, placed in semisolid medium and treated with AG1478 (10 μM) or DMSO control for 24 hours. Real time RT-PCR was used to measure expression of Notch1 and p53. Analysis of c-Fos (as a measure of response to EGFR inhibition) and keratin 1 expression in these tumors is shown in supplemental Fig. 6C.

Fig. 7

Fig. 7. Enhanced apoptosis in squamous carcinoma cells by concomitant suppression of EGFR and Notch signaling

A : SCC13 and SCCO28 cells were treated with DMSO, AG1478 (5 μM), DAPT (20 μM) alone or in combination for 72 h. Cells were analyzed by TUNEL assays and flow cytometry. Values represent the average percentage of apoptotic cells from 3 independent experiments ±SD. B : SCC13 and SCCO28 cells, treated as in previous panel, were analyzed for expression of the pro-apoptotic gene Bim1 by RT-PCR. C : SCC formation was induced in nude mice by subcutaneous injections of SCCO28 cells expressing MAM51 or GFP control (black and white bars, respectively). To minimize individual animal variations, each mouse was injected in parallel, on the right and left flank respectively, with the two kinds of cells. Four weeks after injections, the tumor bearing mice were treated three times, every other day, with AG1478 (1 mg/animal, mice A1, A2, A3). RNA isolated from tumor tissues were analyzed by real time RT-PCR for expression of the indicated genes. D : Frozen sections from the same surgically excised tumors as in the previous panel were analyzed by TUNEL labeling. The fraction of TUNEL positive cells was determined in four different microscopy fields per tumor, using LPLab software. Average percentage of apoptotic cells ± SD is represented. The statistical significance of the observed differences was calculated by Ttest (P<0.05).

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References

    1. Lefort K, Dotto GP. Notch signaling in the integrated control of keratinocyte growth/differentiation and tumor suppression. Semin Cancer Biol. 2004;14:374–386. - PubMed
    1. Lefort K, et al. Notch1 is a p53 target gene involved in human keratinocyte tumor suppression through negative regulation of ROCK1/2 and MRCKalpha kinases. Genes Dev. 2007;21:562–577. - PMC - PubMed
    1. Artavanis-Tsakonas S, Rand MD, Lake RJ. Notch signaling: cell fate control and signal integration in development. Science (New York, NY) 1999;284:770–776. - PubMed
    1. Bray SJ. Notch signalling: a simple pathway becomes complex. Nat Rev Mol Cell Biol. 2006;7:678–689. - PubMed
    1. Weng AP, et al. Growth suppression of pre-T acute lymphoblastic leukemia cells by inhibition of notch signaling. Mol Cell Biol. 2003;23:655–664. - PMC - PubMed

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