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

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.

section_content: The Notch1 gene plays 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 signaling, we identified Epidermal Growth Factor Receptor (EGFR) as a key negative regulator of Notch1 gene expression in normal human keratinocytes, intact epidermis and skin squamous cell carcinomas (SCCs). The underlying mechanism for negative control of the Notch1 gene in the 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 signaling 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 novel role of EGFR signaling in negative regulation of Notch1 gene transcription, of potential relevance for novel combinatory approaches of cancer therapy. 

Cell growth and differentiation are controlled by a complex interplay of signaling pathways functioning in an "integrated", rather than sequential or parallel fashion. Chemical Genetics is based on the principle of using small molecular weight compounds to abrogate or enhance specific regulatory pathways, providing a powerful approach to analyze complex regulatory systems. In the present study we utilized this approach to probe into the signaling network involved in control of Notch1 gene expression and function in human keratinocytes, skin and tumors. Notch signaling plays a key role in promoting keratinocyte differentiation and suppressing keratinocyte-derived tumors 1, 2. Notch receptors, with Notch1 and 2 being the main forms expressed in keratinocytes, are processed by a Ca 2+ -dependent protease in the Golgi prior to transport to the cell membrane. Upon interaction with transmembrane ligands (Jagged1 and 2 or Delta1-4) expressed on the surface of neighboring cells, Notch receptors are activated by consecutive cleavage by an ADAM metalloprotease and a presenilin/-secretase complex. The resulting Notch intracellular domain (ICN) translocates to the nucleus where it associates with the DNA binding protein CSL (CBF-1 or RBP-J in mammalian cells, converting it from a repressor into an activator of transcription 3. Binding of a second ancillary protein, Mastermind-like 1 (MAML1) or related family members, is required for sustained levels of Notch/CSL-dependent transcriptional activation (through recruitment of further transcription co-activators such as p300) 4. 

A 51 amino acid peptide (MAM51) corresponding to the amino terminal region (aa 13-74) of the MAML1 protein competes for MAML1 binding to the Notch/CSL complex, thereby preventing downstream transcription 5. We recently showed that, in primary human keratinocytes, suppression of Notch signaling by this approach results in a lesser commitment to differentiation, expansion of stem cell populations and dramatically increased susceptibility to ras-induced oncogenic conversion 2. Similar effects were observed after pharmacological suppression of endogenous Notch activity by a -secretase inhibitor 2. These findings are likely to be of clinical significance, as Notch1 gene expression and activity are substantially down-modulated in keratinocyte cancer cell lines and tumors, with expression of this gene being under positive p53 control in these cells 2, 6. 

EGFR is among the most intensely studied and well understood determinants of epithelial cell proliferation, and EGFR inhibitors have surfaced as an outstanding example of rational-based drug design for tumors 7. EGFR signaling is likely to function in the keratinocyte proliferative compartment of the epidermis as a "built-in" mechanism to maintain self renewal and, at the same time, suppress differentiation, in contrast to the upper layers where this pathway is down-modulated. In fact, abrogation of EGFR/ERK signaling in proliferating keratinocytes, by either chemical or genetic manipulations, induces differentiation, while sustained activation of this pathway, under conditions where it is normally down modulated, suppresses differentiation 8, 9. This has potentially important implications for keratinocyte-derived tumors, where EGFR signaling is persistently activated and promotes proliferation 10. 

At the biochemical level, little is known on the link between EGFR/ERK pathways and control of differentiation. Here we report a novel role of this pathway in negative regulation of Notch1 gene transcription in both normally proliferating keratinocytes and cancer, which impinges on control of differentiation as well as apoptosis.
section_name: Abstract

section_name: Results

section_content: Little is known of pathways involved in upstream control of Notch1 gene expression and activity in keratinocytes, and mammalian cells in general. To address this issue we undertook a chemical genetics approach. Rather than screening a large collection of unknown chemicals, we chose a library of 489 compounds, approved by the Food and Drug Administration (FDA) and of established target selectivity, using a Luciferase Notch/CSL-responsive reporter as a read-out. The negative regulators of Notch signaling identified by this screen included inhibitors of metalloproteases (MMP) and -secretase, which are required for endogenous Notch activation 4, confirming the validity of the assay (suppl. Table I ). Statistical analysis of the results pointed to a number of other candidate pathways. In particular, the most significant compounds to induce Notch activity were kinase inhibitors that target components of signaling networks connected with EGFR signaling, which was of special interest, given the relevance of this pathway in keratinocytes and cancer 11, 12. 

To validate the findings of our screen, we compared the effects of EGFR inhibition and stimulation on endogenous Notch signaling in human primary keratinocytes. A dose-response of human primary keratinocytes to the EGFR inhibitor AG1478 was determined, on the basis of down-modulation of phospho-EGFR, -ERK1/2, -Jun and -Elk levels, as well as c-Fos expression (Supplemental Fig. 1 ). At the same doses, there was induction of the "canonical" Notch target genes Hes1, Hes5 and Herp1, while, conversely, EGF treatment suppressed expression of these genes (Fig. 1A ). In parallel with this effect, Notch1 mRNA levels were increased by EGFR inhibition, while they were down-regulated by EGF treatment (Fig. 1B ). Consistent with a transcriptional mechanism, no increase of Notch1 mRNA stability was observed in EGFR inhibitor-treated cells after Actinomycin D treatment (data not shown). The results were confirmed at the protein level, by immunoblotting of AG1478-and EGF-treated keratinocytes with antibodies against total and cytoplasmic activated forms of Notch1 as well as Hes1 (Fig. 1C ). Effects similar to those of AG1478 were also elicited by Tarceva, an EGFR inhibitor approved for clinical use (Collins&Workman, Nat Chem Biol 2006) (Supplemental Fig. 2 ). 

Besides chemical inhibition, up-regulation of Notch1 activity and expression were also observed after knockdown of EGFR expression by transfection of keratinocytes with specific siRNAs (Fig. 1D ). Unlike Notch1, Notch2 expression was modulated by EGFR signaling at the mRNA but not protein level (Suppl. Fig. 3A ; Fig. 1C ), while no consistent changes were found in expression of the Notch ligands Jagged 1 and Delta like 1 (Suppl. Fig. 3B, C ). expression is only an indirect consequence of these events. However, treatment of keratinocytes with TNF- at pro-apoptotic concentrations had no effects on levels of Notch1 expression, which was also not affected by suppression of keratinocyte growth by TGF-ß treatment (Supplemental Fig. 2 ) or, as we recently reported, increased expression of cyclin/CDK inhibitors (ANNA's paper). 

The ERK1/2 kinases and the AP-1 transcription complex function as downstream effectors of EGFR activation 11. Induction of Notch1 gene expression similar to that caused by EGFR suppression was observed after siRNA-mediated knockdown of the MEK1 and ERK1 genes while, consistent with their proposed distinct function in keratinocytes (Khavari Cancer Res. 2004 where they show that Only Mek1, however, recapitulated Ras/Raf effects in increasing proliferation and integrin expression while suppressing differentiation, which are impacts characteristic of epidermal neoplasia. ), knockdown of MEK2 or ERK2 had no such effect (Fig. 1E ). 

In contrast to MEK1 and ERK1, no increase of Notch1 expression, or even suppression, was also observed after knock-down and/or pharmacological inhibition of the p38 and JNK kinases, Akt and PKA (Fig. 1E and Supplemental Fig. 2 ). 

Induction of Notch1 expression similar to that caused by EGFR and ERK suppression occurred also after knockdown of c-Jun and c-Fos, two key AP-1 family members (Fig. 1 F, G ). Even in this case, the effects were specific, as they were not observed after knockdown of other AP-1 family members like JunB, Jun D and Fra1, nor of Elk-1, a transcription factor which is activated by EGFR activation though a separate mechanism from AP1 (Friday&Adjei, 2008 Clin Cancer Res. ) (Fig. 1F ).
section_name: Negative regulation of Notch1 gene expression by EGFR/ERK signaling.

section_content: We and others recently showed that the Notch1 gene is a direct transcriptional target of p53 in keratinocytes 2, 6 ADD MANDINOVA. Consistent with these previous results, our chemical screen pointed to a p53 inhibitor, pifithrin, as a negative regulator of Notch signaling (suppl. Table I ), a finding which we directly confirmed by treating keratinocytes with this compound (data not shown). We therefore surmised that a p53-dependent mechanism may underlie up-regulation of Notch1 expression by EGFR suppression. To test this possibility, p53 expression was suppressed in primary keratinocytes by siRNA knock-down. This resulted in reduced levels of Notch1 expression already under basal conditions and, much more substantially, in response to EGFR knock-down (Fig. 2A ). Consistent with a p53-dependent transcriptional control mechanism, luciferase reporter activity of a 2. 4 kbp Notch1 promoter region containing p53 binding sites (but not of a shorter region lacking these sites) was induced in HKCs after EGFR inhibition, with such induction being abrogated by p53 knock-down (Fig. 2B ). Endogenous p53 activity, as assessed by expression of well-established target genes, p21 WAF1/Cip1 and Gadd45 13, was induced as a consequence of EGFR inhibition (Fig. 2C ). There was also a substantial increase of Mdm2, a negative regulator of p53 stability and itself a p53 target gene 13 (Fig. 2D ). Consistent with the negative feedback loop between p53 and Mdm2 protein expression, induction of p53 protein expression by AG1478 became much more evident in cells concomitantly treated with Nutlin, an Mdm2 inhibitor 14 (Fig. 2E ). 

Emerging evidence points to the importance of control of p53 activity by transcription of this gene ( 15-17 and refs. therein). Consistent with this possibility, real time RT-PCR analysis showed that p53 mRNA levels were significantly increased as a consequence of EGFR inhibition while, conversely, were reduced by EGF treatment (Fig. 2F ). Previous work with mouse embryonic fibroblasts indicated that the p53 gene can be a direct target of c-Jun-mediated transcriptional suppression 18. In agreement with this conclusion, chromatin immuno-precipitation experiments showed specific binding of the endogenous c-Jun protein to a predicted AP-1 binding region of the p53 promoter in control but not EGFR-inhibitor-treated cells (Fig. 2G ). 

Functionally, p53 promoter activity (Fig. 2H ) as well as endogenous p53 RNA expression (Fig. 2I ) was induced by siRNA-mediated knockdown of c-Jun, which, as shown before, induces Notch1 expression. A link between the two was demonstrated by the fact that induction of Notch1 expression by c-Jun knock-down was blocked by the concomitant down-modulation of p53 expression (Fig. 2J ).
section_name: Modulation of Notch1 gene transcription by EGFR signaling through p53.

section_content: EGFR signaling provides a break to differentiation, while increased Notch activity promotes this process 1. Real time RT-PCR as well as immunoblot analysis showed that down-modulation of EGFR signaling, by either transfection with siRNAs against EGFR or AG1478 treatment, induced expression of several terminal differentiation markers in keratinocytes, including Keratin1 and 10 and Involucrin (supplemental Fig. 4 and data not shown). To assess whether induction of differentiation by EGFR suppression is due to up-regulation of Notch signaling, primary human keratinocytes were infected with a retroviral vector expressing a 51 amino acid peptide (MAM51) that competes for MAML1 binding to the Notch/CBF-1 complex, thereby preventing downstream transcription 5. Treatment of MAM51expressing keratinocytes with AG1478 caused a similar induction of Notch1 expression as control cells; by contrast, induction of Hes1 and differentiation markers was suppressed (Fig. 3A ). Chemical inhibitors of -secretase activity like DAPT suppress proteolysis-dependent activation of endogenous Notch receptors 19. As with MAM51 expression, treatment of primary keratinocytes with DAPT counteracted induction of Hes1 and differentiation marker expression caused by EGFR downmodulation (Fig. 3B ). Similar counteracting effects were also observed after siRNAmediated knock-down of Notch1 and p53 expression, indicating that up-regulation of these genes by EGFR inhibition is responsible for the observed induction of differentiation (Fig. 3C ). A more indirect downstream mechanism, like that reported in Drosophila with EGFR signaling suppressing Groucho-dependent transcription 20, is unlikely in our system. In fact, consistent with our previous findings, EGFR activation exerted no counteracting effects on induction of differentiation by activated Notch1 (Fig. 3D ) and increased Hes1 expression, a mammalian Groucho effector 21, suppressed rather then induced differentiation (Fig. 3E ), confirming what previously observed for Hes1 effects in mouse primary keratinocytes 31. 

Growth/differentiation control of keratinocytes in culture is likely to differ in significant aspects from control of these cells in intact skin. For further validation of our findings, we resorted to several complementary approaches. For the first, mice with a GFP reporter for Notch activity were injected with the EGFR inhibitor AG1478. 

Immunofluorescence analysis showed significantly elevated GFP expression in the epidermis of the AG1478 treated mice versus the control (Fig. 4A ). 

For confirmation and quantification of the results, the epidermis of these mice was separated from the underlying dermis by a brief heat treatment, followed by total RNA preparation and real time RT-PCR analysis. This confirmed increased GFP expression, which paralleled increased expression of the endogenous Notch1, p53 and Keratin 1 genes (Fig. 4B, C ). As a second approach, the epidermis of homozygous mice for a hypomorphic EGFR mutation was analyzed, in parallel with heterozygous littermates. Even in this case, decreased EGFR activity was found to result in increased p53 and Notch1 expression (Fig. 4D ). 

In senescence driven skin aging and data not shown). Similar results were obtained with an organ culture system of freshly excised human skin, using an optimized method that allows maintenance of viable tissue with no sign of degeneration and/or altered differentiation for up to 7 days (our unpublished observations). 

Immunohistochemical as well as real time RT-PCR analysis indicated that, even in these conditions, EGFR inhibition caused a parallel induction of Notch1 activity and expression, and differentiation in keratinocytes, together with p53 and p21 WAF1/Cip1 (Fig. 4F, Supplemental Fig. 5 ). Using this approach, we tested whether the increase in keratinocyte differentiation is Notch dependent. For this, human skin cultures were treated with AG1478 plus/minus DAPT. As shown in Fig. 4G, induction of Keratin 1

and Involucrin expression by EGFR inhibition was counteracted by the concomitant treatment with the Notch / -secretase inhibitor, while, as expected, induction of the Notch1 gene itself, or of p53, was unaffected or even increased.
section_name: EGFR-p53-Notch control of differentiation in primary keratinocytes and intact skin.

section_content: To assess whether the p53/Notch regulatory loop discovered here applies to conditions where increased EGFR signaling has been causally linked with cancer development, we analyzed transgenic mice expressing a constitutive active form of the EGFR/Ras adaptor protein SOS under control of a keratin 5 promoter (K5-SOS-F). These mice develop spontaneous skin tumors strictly dependent on the presence of functional EGFR 22. In K5-SOS-F transgenics with a concomitant keratinocytespecific deletion of the c-Jun gene, skin tumor development is impaired, correlating with reduced EGFR expression and increased differentiation 23. The present results suggested that EGFR/c-Jun regulation of p53 and Notch1 expression may also be involved. In fact, real time RT-PCR analysis showed significantly higher levels of Notch1 and p53 expression in the small tumors formed by K5-SOS-F transgenic mice with epidermal deletion of the c-Jun gene (c-Jun ep SOS + ) relative to tumors formed in K5-SOS-F transgenics with the intact c-Jun gene (c-Jun f/f SOS + ) (Fig. 5A ). These data were confirmed at the protein level by immunoblot analysis of a separate set of tumors, as well as by immunofluorescence for Notch1 expression (Fig. 5B, C ). 

To assess whether similar EGFR regulation of Notch1 expression applies to human cancer, keratinocyte-derived SCC cells (SCCO28, SCC12 and SCC13) with wild type p53 (http://www. sanger. ac. uk/genetics/CGP/CellLines/) were treated with EGFR inhibitor. Besides mutations, p53 activity can also be reduced in tumors as a consequence of decreased p53 gene transcription 17, [24] [25] [26] [27]. Consistent with this mode of regulation, EGFR inhibition of SCC cells induced expression of the p53 gene as well as of p21 WAF1/Cip1, indicative of increased p53 activity (Fig. 6 A, B ). This was paralleled by a substantial increase of Notch1 mRNA and protein levels and differentiation markers (Fig. 6C,D: Supplemental Fig. 6A ). As with primary keratinocytes, p53 knockdown experiments showed that even in cancer cells, induction of Notch1 expression by EGFR-inhibition is p53-dependent (Fig. 6E ). 

Cancer cell lines can differ substantially in their control mechanisms from cells in primary tumors. Therefore, as a further validation of our findings, the same organ culture system described above for intact skin was adapted to the analysis of clinically occurring SCCs freshly excised from patients. The dissected more homogeneous parts of tumors were cut into small pieces of the same size (2x2 mm), and placed into multi-well dishes as for skin organ cultures. In five independent tumors, EGFR inhibition resulted in reduction of c-Fos expression, indicative of EGFR signaling suppression, and concomitant induction of Notch1, p53 and Keratin 1 (Fig. 6F ; Supplemental Fig. 6C ). In four other tumors no such effects were observed, consistent, in two cases, with resistance of EGFR inhibition (as assessed by no decrease in c-Fos expression) and, in the other two, undetectable p53 expression or activity (data not shown).
section_name: Inhibition of EGFR signaling in cancer cells induces Notch1 gene expression through p53.

section_content: As with primary keratinocytes, even in SCC cells inhibition of EGFR signaling caused up-regulation of differentiation markers expression, through a Notch dependent mechanism (Supplemental Fig. 6A, B ). We have recently found that Notch-dependent differentiation of keratinocytes render these cells more resistant to apoptosis (REF Mandinova et al. ). Thus, an attractive possibility was that suppression of Notch signaling, while suppressing the pro-differentiation effects of EGFR inhibitors, may synergize with these compounds in triggering apoptosis. To assess this possibility, SCC cells were treated with DAPT plus-minus EGFR inhibitor. As shown in Fig. 7A, the concomitant treatment led to a substantial increase of apoptosis. These findings were paralleled by a synergistic induction of Bim1 expression (Fig. 7B ), a proapoptotic Bcl2 family member that has been recently implicated in the response of cancer cells to EGFR inhibitors 28. 

To further validate the relevance of these findings for the behavior of cancer in vivo, immune-compromised mice were injected with SCC cells expressing the Notch inhibitory MAM51 peptide, in parallel with control cells. After formation of sizable tumors (4 weeks after injections), mice were treated with AG1478 for a week. RT-PCR analysis of tumor RNAs showed substantially higher levels of Hes1 and differentiation marker expression in tumors formed by control than MAM51-expressing cells, while levels of Bim1 were oppositely regulated (Fig. 7C ). This was paralleled by a higher apoptotic fraction in tumors with suppressed Notch signaling (Fig. 7D ).
section_name: Inhibition of Notch signaling in cancer cells suppresses differentiation induced by EGFR suppression while it synergizes for apoptosis.

section_content: Loss-and gain-of function experiments in mice as well as work with human keratinocytes have established that EGFR signaling plays a key role in positive control of keratinocyte growth potential and carcinogenesis 10. A similar role has been found for downstream effectors of this pathway at the level of transcription, like c-Jun 9. Besides enhancing proliferation, we have shown here that EGFR signaling plays a significant role in suppressing differentiation through negative regulation of Notch1 gene expression and activity. This mechanism is likely to provide a break for the commitment to differentiation of keratinocytes in the basal proliferative compartment of the epidermis as well as in cancer, where EGFR signaling is characteristically elevated 10. 

In the upper epidermal layers, EGFR signaling is normally down-modulated and therefore ceases to be relevant. In fact, differentiation of keratinocytes as they migrate to the upper epidermal layers is induced by multiple EGFR-unrelated events including, most notably, loss of integrin-mediated adhesion to the matrix and establishment of cadherin-dependent cell-cell adhesion 29. This is consistent with control of the Notch1 gene by EGFR signaling -via p53 -being relevant for the behavior of proliferating keratinocytes in normal skin and cancer, while additional multiple mechanisms are responsible for the increase of Notch signaling in normally differentiating keratinocytes of the upper epidermal layers 1. 

We have shown that suppression of the EGFR signaling cascade leads to upregulation of Notch1 gene expression through a mechanism involving transcriptional up-regulation of the p53 gene. Previous studies pointed to NF-B control of p53 gene expression 30, which could be of relevance to the present situation, as NF-B activity is induced in keratinocytes with differentiation 31, while its suppression promotes tumor development 32. However, expression of NF-B responsive genes, as an indication of endogenous activity, is not induced in keratinocytes by EGFR suppression (data not shown). As an alternative mechanism, we have found that control by AP-1 family members is involved. AP-1 is a heterodimeric DNA-binding complex formed by proteins of the c-Jun and c-Fos families, which differ in their ability to activate target genes and can also function in an inhibitory fashion 33. 

Previous work with mouse embryo fibroblasts has indicated that c-Jun can be a direct negative regulator of p53 gene expression 18. Consistent with this conclusion, we have found that endogenous c-Jun binds to the p53 promoter in both normal and SCCderived keratinocytes, and that expression of the p53 gene in these cells is enhanced by c-Jun knock-down. Notch1 expression is also induced by c-Jun knockdown, in a p53-dependent fashion. This concomitant mode of regulation of p53/Notch1 expression by EGFR signaling was further validated by our findings with organ cultures of intact human skin and SCCs, as well as a mouse model of skin cancer formation dependent on EGFR and c-Jun function. 

EGFR has become an important target of cancer drug design, and several selective EGFR inhibitors have now been approved for clinical use. Recent data have highlighted the fact that inhibitors directed to critical receptors like EGFR utilize the cell death pathway for inducing tumor regression 28. However, the exact molecular mechanisms underlying sensitivity and resistance of tumor cells to EGFR inhibitionincluding the contribution of other integrated pathways like the ones we have identified here -remain to be elucidated 34. Importantly, suppression of Notch signaling in squamous carcninoma cells counteracts the differentiation inducing effects of EGFR inhibitors, while, at the same time, synergizing with these compounds in induction of apoptosis. Other ongoing studies in the laboratory indicate that the enhancing effects of Notch suppression on apoptosis may extend also to lung cancer cells. This suggests an attractive new avenue of combination approaches for cancer therapy that may enhance the potency of EGFR inhibitory agents on tumors, while at the same time ameliorating their well known toxic effects on the skin which have been attributed, at least in part, to aberrant differentiation 12.
section_name: Discussion

section_content: Cell culture and viruses. 

Culturing of primary human keratinocytes and SCC 12, 13 and 028 cells, and infection with the MSCV-MAM51 5 and control retrovirus were as previously reported 2. Adenoviruses for Notch1, Hes1 and GFP and adenoviral infections were previously described 31. NCI-H1299 and NCI-H1299 were cultured in RPMI medium (Invitrogen) supplemented with 10% bovine serum. Chemical inhibitors, AG1478

(LC Labs), Nutlin (Calbiochem), DAPT (Calbiochem), ERK inhibitory peptide-cell permeable (Calbiochem) were dissolved in DMSO and applied at the indicated concentrations. For knock down experiments, cells were transfected as described 31 with validated stealth siRNAs for human EGFR, p53, and c-Jun in parallel with corresponding Stealth siRNA controls (Invitrogen), or ERK1, ERK2 and Notch1 (GeneGlobe, Qiagen) and analyzed 48-72 hours after transfection.
section_name: Methods

section_content: Conditions for real time PCR analysis, chromatin immunoprecipitation Chip), immunoblotting and immunofluorescence were as previously described 2. The list of gene-specific primers is provided in Supplemental Table II. We used the following antibodies: Notch1 (Santa Cruz, C-20), activated Notch1 (Cell Signaling), Hes1 (Chemicon AB5702), Keratin1 (Babco AF87), Involucrin (Babco PRB140C), EGFR (Cell Signaling), p53 (Cell Signaling), MDM2 (Cell Signaling), Integrin 4 (Santa Cruz), -Tubulin (Sigma GTU87), for immunoblotting for mouse proteins: Notch1 (Pharmingen), p53 (Novacastra), c-Jun (Transduction Labs) and Actin (Sigma), c-Jun for Chip assays (Santa Cruz, H79).
section_name: Quantitative real time RT PCR, chromatin immunoprecipitation and immunodetection techniques.

section_content: Discarded human skin samples from abdominoplasty procedures were obtained from the Centre Hospitalier -Universitaire Vaudois (Lausanne, Switzerland) under patients' agreement and institutional approval. Skin samples, sterilized in 70% ethanol and cut, after removal of subcutaneous fat, into 1x1cm pieces, were placed in keratinocyte serum-free medium (KSF, GIBCO-BRL) supplemented with epidermal growth factor (EGF) and bovine pituitary extract (BPE), in 0. 25% agar (Sigma). The epidermis was maintained at the air-medium interface. For RNA collection, skin samples were placed in preheated PBS at 60 °C for 45 seconds, then chilled (on ice) in 0. 1M PBS for 1 minute, followed by mechanical separation of epidermis and dermis. The epidermis was homogenized in TRI Reagent (Sigma) for RNA preparation. Human SCC samples were obtained as discarded material from Mohs micrographic surgery at Massachusetts General Hospital (Boston, MA) with patients' and institutional approvals. Tumor samples were sterilized in 70% ethanol, cut into pieces of approximately 2x2 mm and placed in semi-solid medium similarly to skin organ cultures.
section_name: Organ cultures

section_content: For in vivo tumorigenicity assays, control and MAM51 expressing SCCO28 cells were brought into suspension, admixed with Matrigel (BD Biosciences), and injected (5x10 6 cells/injection) subcutaneously in 8 weeks old female athymic nude mice. Four weeks later animals were treated three times (every other day) with AG1478 (1 mg/animal, dissolved in 200 l 50% DMSO:DMEM) or DMSO vehicle control by i. p. injections. Mice were sacrificed 2 days after the last treatment and tumors processed for RNA preparation and analysis.
section_name: Tumorigenicity assays

section_content: Cells were trypsinized, recovered by centrifugation at 300 g and fixed in 2% paraformaldehyde in PBS for 16 h. Permeabilization and enzymatic labeling with TMR red-conjugated-dUTP were performed according to the manufacturer's protocol (Roche, IN). The percentage of cells that incorporated the fluorescence-conjugated dUTP was determined by flow cytometry. TUNEL assay on histological sections was analyzed with fluorescent microscopy and IPLab software.
section_name: TUNEL Assays

section_name: Acknowledgments.

section_content: Drs. Jim Follen and Caroline Shamu of the Harvard Institute for Chemistry and Cell Biology and Nichola Tolliday from the Broad Institute (Harvard/MIT, Cambridge, MA ) for assistance with screening, Drs. W. Austen ( MGH, Boston, MA ) and W. Raffoul ( U. of Lausanne ) for human skin material, Drs. Jeff Settleman and Ultan McDermott ( MGH, Boston, MA ) for the lung cancer cell lines, Dr. Rainer Zenz for mouse skin tumor samples, Vikram Rajashakera for technical help, and Drs. C. Brisken and C. Missero for careful reading of the manuscript,. This work was supported by NIH Grants AR39190, CA16038 and CA73796, the Swiss National Foundation, a grant from the European Union ( Epistem, Sixth Framework Program, LSHB-CT-2005-019067 ) and, in part, by the Cutaneous Biology Research Center through the Massachusetts General Hospital / Shiseido Co. Ltd. Agreement.
section_name: We thank