Cancer Research Infection and Cancer: Biology, Therapeutics, and Prevention
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
Cancer Research 67, 7654-7664, August 15, 2007. doi: 10.1158/0008-5472.CAN-06-4415
© 2007 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Brouxhon, S.
Right arrow Articles by Pentland, A. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Brouxhon, S.
Right arrow Articles by Pentland, A. P.

Cell, Tumor, and Stem Cell Biology

Sequential Down-regulation of E-Cadherin with Squamous Cell Carcinoma Progression: Loss of E-Cadherin via a Prostaglandin E2-EP2–Dependent Posttranslational Mechanism

Sabine Brouxhon1,3, Stephanos Kyrkanides2,4, M. Kerry O'Banion2, Renee Johnson2, David A. Pearce5, Gina M. Centola4, Jen-nie H. Miller4, Kieran H. McGrath3, Brandon Erdle3, Glynis Scott3, Sandra Schneider1, JoAnne VanBuskirk3 and Alice P. Pentland3

Departments of 1 Emergency Medicine, 2 Neurobiology and Anatomy, and 3 Dermatology; 4 Eastman Dental Center; and 5 Departments of Biochemistry/Biophysics, Neurology and Center for Aging and Developmental Biology, University of Rochester School of Medicine and Dentistry, Rochester, New York

Requests for reprints: Sabine Brouxhon, Department of Emergency Medicine, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Box 670, Rochester, NY 14642. Phone: 585-275-2714; Fax: 585-473-3516; E-mail: Sabine_Brouxhon{at}urmc.rochester.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The incidence of skin cancer is on the rise, with over 1 million new cases yearly. Although it is known that squamous cell cancers (SCC) are caused by UV light, the mechanism(s) involved remains poorly understood. In vitro studies with epithelial cells or reports examining malignant skin lesions suggest that loss of E-cadherin–mediated cell-cell contacts may contribute to SCCs. Other studies show a pivotal role for cyclooxygenase-dependent prostaglandin E2 (PGE2) synthesis in this process. Using chronically UV-irradiated SKH-1 mice, we show a sequential loss of E-cadherin–mediated cell-cell contacts as lesions progress from dysplasia to SCCs. This E-cadherin down-regulation was also evident after acute UV exposure in vivo. In both chronic and acute UV injury, E-cadherin levels declined at a time when epidermal PGE2 synthesis was enhanced. Inhibition of PGE2 synthesis by indomethacin in vitro, targeted deletion of EP2 in primary mouse keratinocyte (PMK) cultures or deletion of the EP2 receptor in vivo abrogated this UV-induced E-cadherin down-regulation. In contrast, addition of PGE2 or the EP2 receptor agonist butaprost to PMK produced a dose- and time-dependent decrease in E-cadherin. We also show that UV irradiation, via the PGE2-EP2 signaling pathway, may initiate tumorigenesis in keratinocytes by down-regulating E-cadherin–mediated cell-cell contacts through its mobilization away from the cell membrane, internalization into the cytoplasm, and shuttling through the lysosome and proteasome degradation pathways. Further understanding of how UV-PGE2-EP2 down-regulates E-cadherin may lead to novel chemopreventative strategies for the treatment of skin and other epithelial cancers. [Cancer Res 2007;67(16):7654–64]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The incidence of skin cancer is on the rise, with over 1 million new cases of basal and squamous cell carcinoma (SCC) each year (1). These skin cancers are caused by chronic exposure to UV light and pose a major health care challenge due to the morbidity, mortality, and medical costs associated with tumors and their treatment. An improved understanding of the cellular and molecular changes following UV insults that lead to the generation of these cancers will provide new opportunities for their treatment.

Profound alterations in the glycoprotein E-cadherin, which serves to promote the establishment of homotypic cell-cell adhesions, have been associated with the development and progression of breast, intestinal, and other epithelial cancers (2). It is now well established that E-cadherin loss is causally involved in the progression of pancreatic ß-cell carcinomas (3). Moreover, several groups have shown that defective E-cadherin function can render noninvasive cells invasive, whereas reestablishing functional E-cadherin complexes in tumorigenic cell lines reverts an invasive mesenchymal phenotype to a benign epithelial phenotype (4–6). In skin, in vitro work and studies using human malignant skin lesions suggest that decreased E-cadherin function plays a critical role in SCC progression (7–10). However, exposure to chronic UV irradiation (UVR) remains the largest risk factor associated with the development of SCCs, and these prior studies did not determine the mechanism(s) by which E-cadherin expression is temporally regulated by acute or chronic doses of UVR in vivo. Instead, they only focused on the role of the E-cadherin adhesion system in disease states once tumors have already formed. Moreover, whether E-cadherin is modulated by UVR using similar molecular mechanisms as that found in other malignancies is unknown.

Recent studies support the hypothesis that loss of E-cadherin and enhanced prostaglandin E2 (PGE2) synthesis independently play an important role in the pathogenesis of various epithelial tumors. However, there has been no direct evidence showing a link between PGE2 and E-cadherin loss of expression after UVR in skin. PGE2 is the most abundant prostaglandin produced by human keratinocytes after irradiation injury and is a powerful regulator of keratinocyte proliferation (11). Many epithelial cancers produce large amounts of PGE2, with levels rising dramatically as cells progress to an invasive phenotype (12–14). In later stages of carcinogenesis, PGE2 can directly stimulate tumor growth, migration, and invasion (15–17). A role for PGE2 in modulating cell shape and morphology has also been described (18). Moreover, studies conducted in mice suggest that PGE2 signaling by EP2 receptor activation is important in the development and progression of intestinal, breast, and skin cancers (19–23). However, despite demonstration of a causative role for UV-induced PGE2-EP receptor activation in this neoplastic transformation, many aspects of the underlying cellular and molecular mechanisms have yet to be elucidated.

In this study, we investigated the role of E-cadherin in UV-induced SCC progression and after acute single doses of UVR in vivo and in vitro. We also examined whether UVR contributes to this dysregulation of E-cadherin expression via a PGE2-EP2 receptor-dependent mechanism.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice. Outbred SKH-1 mice are the standard strain used for photocarcinogenesis work (24). Six- to 7-week-old wild-type (WT) and EP2–/– mice (generated by Dr. Richard Breyer, Division of Nephrology and Departments of Medicine and Pharmacology, Vanderbilt University, Nashville, TN, as described previously; ref. 22) weighing between 25 and 30 g were used. The mice were housed five per cage under constant humidity and temperature with 12-h light/12-h dark cycles. They were allowed access to water and standard mouse feed ad libitum and were monitored daily. All experimental procedures were approved by the Institutional Laboratory Animal Care and Use Committee of the University of Rochester Medical Center.

UV irradiation. The dorsal skin of WT and EP2–/– knockout mice was exposed to UVR in groups of two or three using a bank of four UVA Sun 340 sunlamps as described previously (22, 25). Briefly, UVA Sun 340 sunlamps emit UV between 295 and 390 nm (includes both UVA and UVB wavelengths). Lamp emission closely resembles the UV spectrum of sunshine through the mid-UVA range (22). Lamp output was measured by an IL1700 light meter (International Light) using an SED 240 probe for measurement of the UVB portion of the lamp spectrum. The SED 240 probe detects wavelengths from 255 to 320 nm. Doses stated represent this portion of the lamp output only. The mice were exposed to UV at a distance of 15 inches. Animals were irradiated acutely with 180 mJ/cm2 UVR. In the chronic irradiation protocol, animals were irradiated initially with 120 mJ/cm2 of UVR, thrice weekly, increasing the exposure dose 10% each week for 15 weeks. Tumor development was observed over the subsequent 15 weeks. The maximum length of exposure was 300 min. The cumulative UVB dose was 12 J/cm2. The UVA cumulative dose was 658 J/cm2.

For in vitro experiments, confluent WT and EP2–/– primary mouse keratinocytes (PMK) were exposed to UVR through a Schott WG 295 glass filter (BES Optics) and FS20 sunlamps (Westinghouse). This light source emits primarily in the UVB spectrum (290–320 nm), with minor output in the UVA range. Cell cultures were exposed to doses of UV between 10 and 60 mJ/cm2. Sham-irradiated cultures (shielded with aluminum foil during UVR) served as controls.

Tumor tissues and morphologic studies. Chronically UV-irradiated epidermis (15 weeks after 15 weeks of chronic UV exposure) was isolated from the dorsum of SKH-1 mice by curettage of flash-frozen whole mouse skin. Efficacy of curettage as a method for removing epidermis was documented by histology. Small and large tumors (SCCs; >2 mm) were isolated using a prechilled scalpel blade. All tissues were placed immediately in cold Falcon tubes and stored at –80°C for further analysis.

Tissue sections and immunohistochemistry. After sacrifice, half of the mouse dorsal skin was snap frozen in liquid nitrogen and stored at –80°C. The remaining tissue was fixed in 10% formalin and embedded in paraffin. Immunolocalization of E-cadherin was done as described previously (26). Briefly, deparaffinized 5-µm sections were washed in PBS, blocked in 4% normal goat serum, and incubated overnight with either anti-E-cadherin (Santa Cruz Biotechnology), anti-Ki67 antibody (Abcam), or an anti-cyclin D1 (Santa Cruz Biotechnology) polyclonal antibody at 4°C. The primary antibody was detected with the Vectastain rabbit avidin-biotin complex kit (Vector Laboratories). Appropriate isotype controls were processed simultaneously. For histologic analysis of Ki67, sections were imaged by bright-field microscopy (Bx51 Olympus) and the number of Ki67-positive cells was quantitated from a minimum of 10 random fields using 40x objective.

Isolation of PMK, cell culture, and treatments. Mice were sacrificed by CO2 inhalation, cleansed with betadine/antimicrobial soap, and rinsed in water. Dorsal/ventral skin was excised and rinsed in PBS containing 10 units/mL penicillin and 10 µg/mL streptomycin (Invitrogen). Subcutaneous fat was removed and the skin was incubated for 60 to 90 min at room temperature in PBS containing 2.5% trypsin (Invitrogen). PMKs were collected by gentle scraping of trypsin-separated epidermal sheets with curved forceps, centrifugation at 1,000 x g for 5 min, and resuspension in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 25 mmol/L HEPES buffer, and antibiotic-antimycotic solutions (100 units/mL penicillin, 25 µg amphotericin B, and 100 µg/mL streptomycin; Invitrogen). Cells were seeded onto collagen-1–precoated (Invitrogen) six-well culture dishes or Lab-Tek chamber slides (Nalge Nunc International) and maintained at 37°C in a humidified atmosphere of 95% air and 5% CO2. Cells were cultured for 48 h until they achieved >90% confluence. For PGE2, indomethacin, or butaprost treatment, cultures were maintained for 24 h in the presence/absence of 3 µg/mL indomethacin with and without UVR. Alternatively, unirradiated cells were cultured in the presence of 10–6 to 10–10 mol/L of PGE2 or 0.01 to 100 ng/mL of butaprost in the presence of 3 µg/mL indomethacin. In all experiments, cells were pretreated with 3 µg/mL indomethacin for 30 min before UVR to block endogenous production of PGE2 in the culture system so that the exact amounts of exogenous PGE2 would be reflected by the amount of PGE2 added. Control cultures consisted of cells incubated in medium without these reagents or in indomethacin alone. An equal volume of drug vehicle (ethanol) also served as a control. After 30 min to 72 h, cells were harvested and processed for Western blotting. Indomethacin was purchased from Sigma. PGE2 and butaprost were purchased from Cayman Chemical. For lysosomal and proteasome inhibitor treatments, PMKs were incubated for 1 h before UVR with the lysosome inhibitor chloroquine (50 µmol/L; Sigma) or the proteasome inhibitor N-acetyl-Leu-Leu-Nle-aldehyde (ALLN; 50 µmol/L; Calbiochem). Twenty-four hours after UVR, cells were lysed and processed for Western immunoblotting.

Fluorescence microscopy. Double immunofluorescence methods were done as described previously (27). Briefly, cells were grown on chamber slides, fixed with 4% formaldehyde, and permeabilized with 0.4% Triton X-100. For the detection of E-cadherin, we used a rabbit anti-E-cadherin antibody coupled with a goat anti-rabbit Alexa Fluor 488 fluorescent secondary antibody (Molecular Probes). Nuclei were visualized by incubating cells with the Hoechst counterstain (Invitrogen). Color fluorescent images were captured separately under 488 nm (green) and 350 nm (blue) wavelengths and processed using Adobe Photoshop software.

Quantitation of prostaglandins. After sacrifice, mouse dorsal skin was snap frozen and the epidermis was curetted directly into ice-cold methanol as described previously (22, 25). Thromboxane B2 (Cayman Chemical) was added to samples for estimation of extraction efficiency and then buffered with 0.1 mol/L NaH2PO4 (pH 4.0), and then individually loaded onto C-18 solid-phase extraction cartridges (Waters) preconditioned with ethanol and H2O at pH 4. After washing with H2O (pH 4.0) and hexane, samples were eluted by gravity with ethyl acetate/1% methanol. The eluant was dried under nitrogen and reconstituted with EIA buffer (Cayman Chemical). The PGE2 levels in the samples were determined using the PGE2 EIA kit (Cayman Chemical). Epidermal protein content was determined, and results were expressed as extracted PGE2/mg tissue. In tissue culture experiments, PGE2 content in conditioned supernatants was determined and then normalized to the protein content of the culture. Full thickness epidermis contains a significant percentage of nonviable stratum corneum, producing lower measured levels of PGE2 content when compared with tissue culture data.

Western blot analysis. Snap-frozen curetted epidermis, small and large tumors, or cultured PMKs were prepared by homogenization in radioimmunoprecipitation assay buffer (0.5% deoxycholate, 0.1% SDS, 1% NP40, 20 mmol/L sodium orthovanadate, 50 mmol/L NaF) containing a 1:100 volume of protease inhibitors (Protease inhibitor cocktail, Sigma). After homogenization, protein concentration was determined using a bicinchoninic acid protein assay kit (Pierce). Aliquots of 20 to 50 µg of total protein were resolved by SDS-PAGE, transferred to nitrocellulose membrane, and probed with respective antibodies. This was followed by incubation with horseradish peroxidase–linked secondary antibodies and visualization of proteins by chemiluminescence (Pierce). Protein loading was normalized by staining with an anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Ambion). Band intensity was quantified using NIH Scion Image and normalized to GAPDH.

Statistical analysis. Comparisons between groups were made using ANOVA. Differences between treatment and control conditions were established by post hoc analysis using the Student-Newman-Keuls or Dunnett's method, as appropriate. Statistical significance is indicated in figures as follows: *, P < 0.05; **, P < 0.01; or ***, P < 0.001.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
E-cadherin is sequentially lost in UV-induced SCC progression and after an acute UV exposure in SKH-1 mice. Because the most important environmental risk factor for SCC is exposure to solar UV radiation, particularly its UVB component, we first wanted to determine if UV-induced SCC progression in mice recapitulates what has been reported in human skin constructs or human SCCs. We therefore examined samples of chronically UV-exposed epidermis and resected small and large tumors from mice that had completed a chronic photocarcinogenesis protocol. Samples were analyzed for E-cadherin expression by Western blotting (Fig. 1A ). A statistically significant decrease in E-cadherin protein with SCC progression was observed (P < 0.0001, ANOVA). Chronically irradiated epidermis and small and large overt tumors contained progressively less E-cadherin protein compared with age-matched controls (53%, 82%, and 99%, respectively).


Figure 1
View larger version (58K):
[in this window]
[in a new window]

  		Figure 1. E-cadherin down-regulation with UV-induced SCC progression and following acute UVR in the epidermis of mice. A, SKH-1 mice were irradiated initially with 180 mJ/cm2 of UVR, thrice weekly, increasing the exposure dose 10% each week for 15 wks. Tumor formation was followed over the subsequent 15 wks (weeks 15–30). At the 30-week time point, small tumors (ST), large tumors >2 mm (T), and 30-wk non–tumor-bearing tissue (Chronic UV) was excised and assessed for E-cadherin levels by Western immunoblotting. E-cadherin levels were significantly different for control (C) versus chronic UV, control versus small tumors, control versus large tumors, and chronic UV versus small tumors as assessed by ANOVA followed by post hoc analysis using the Dunnett's method (all pairwise multiple comparison procedures). ***, P < 0.0001, n = 4 per group. Inset, representative immunoblot of E-cadherin in control, chronic UV, small tumors, and large tumors. E-cadherin protein levels were normalized to GAPDH and quantified by using NIH Scion Image software. Equal loading of protein was verified by GAPDH staining. B, immunostaining of E-cadherin (magnification, x20 to x60) in age-matched control skin, chronic UV-exposed skin, small tumors, and large tumors. High magnifications (x100) are included as insets for the small and large tumors. C, SKH-1 mice were irradiated with an acute 180 mJ/cm2 dose of UVR, and 24 and 72 h later, the epidermis was curetted and processed for Western immunoblotting. Densitometric analysis of band intensity for E-cadherin levels in epidermal curettings of acutely irradiated mice. E-cadherin levels were significantly different for control versus 24 h and control versus 72 h (n = 5 per group). ***, P < 0.001. Inset, representative Western blot for E-cadherin from one of five separate experiments. E-cadherin levels were normalized to GAPDH and quantitated by NIH Scion Image. D, SKH-1 mice were irradiated with an acute 180 mJ/cm2 dose of UVR, and 72 h later, skin sections were resected, formalin fixed, paraffin embedded, and immunostained for E-cadherin by immunohistochemistry. Skin from unirradiated mice. Isotype control is included.

 
We next examined E-cadherin immunostaining by immunohistochemistry and found a disruption of E-cadherin staining in areas of chronic UV-exposed epidermis that exhibited cellular atypia, resembling SCC in situ (Bowen's disease; Fig. 1B). In the small tumors, E-cadherin staining was scattered at sites of cell-cell contacts in small isolated areas, but overall, the staining intensity was markedly reduced from control unirradiated skin. In contrast, large invasive SCCs exhibited a robust inflammatory infiltrate with epithelial cells largely devoid of E-cadherin staining, except at the tumor border edge that abutted normal-appearing epithelium (Fig. 1B).

We have observed a change in the shape and polarity of cells in the basal and stratified layer of the epidermis after an acute UV exposure.6 We therefore examined E-cadherin protein levels following an acute single UV exposure. Mice were exposed to a single 180 mJ/cm2 UV dose and then sacrificed 24 or 72 h afterward. Western blot analyses of epidermal curettings showed a significant UV-induced decrease in E-cadherin protein levels at 24 and 72 h compared with unirradiated controls (P < 0.001; Fig. 1C). Immunohistochemistry of control skin sections revealed a staining pattern for E-cadherin that was most pronounced at points of cell-cell contact throughout the epidermis (Fig. 1D). In contrast, UV-irradiated skin exhibited epidermal thickening and a marked decrease in E-cadherin staining in all areas of the epidermis except the basal layer and cells forming the outermost layer of the outer root sheath of the hair follicle.

Acute and chronic UV-induced E-cadherin loss correlates with heightened PGE2 synthesis in SKH-1 mice. We next examined PGE2 synthesis in the epidermis of SKH-1 mice exposed to acute (180 mJ/cm2) and chronic (15 weeks) doses of UVR. After acute injury, PGE2 synthesis increased at 24 h (control = 91.5 ± 11.0 pg/mg protein; UV = 196.5 ± 8.9 pg/mg protein) and remained slightly above unirradiated controls at the 72-h time point (135.6 ± 22.9 pg/mg protein). When skin samples from animals were examined after chronic UV injury (24 h after the last UV exposure), epidermal PGE2 content increased 6-fold (560 ± 90.88 pg/mg protein) over unirradiated controls. Therefore, at time points when UV-induced E-cadherin protein levels were decreased, PGE2 production was enhanced.

UV-induced E-cadherin loss correlates with enhanced PGE2 synthesis in vitro. To dissect the processes responsible for the changes observed acutely and chronically in vivo, cultured PMKs were isolated from WT SKH-1 mice and exposed to an acute dose and time course of UVR (Fig. 2 ). Exposure of PMK to UVR in doses from 0 to 40 mJ/cm2 caused a significant dose-dependent decrease in E-cadherin protein levels, with a maximal response after exposure to 40 mJ/cm2 (P < 0.001; Fig. 2A). The time course of this effect was studied in cultures exposed to 40 mJ/cm2. A statistically significant time-dependent reduction in E-cadherin protein levels was found at 6, 24, and 72 h (P < 0.001; Fig. 2B). Because the in vivo data presented above suggest a temporal association between UV-induced PGE2 synthesis and E-cadherin loss, PGE2 levels in the supernatants were also assessed by ELISA. As expected, PGE2 analysis showed a statistically significant dose-dependent increase up to the 40 mJ/cm2 dose (P < 0.01; Fig. 2C).


Figure 2
View larger version (13K):
[in this window]
[in a new window]

  		Figure 2. Decreased E-cadherin expression and heightened PGE2 content in PMK after acute UVR in vitro. A, confluent PMKs were treated with UVR at the doses indicated. Cells were harvested 24 h later. Unirradiated PMKs were included as controls. Immunoblot analysis for E-cadherin normalized to GAPDH (NIH Scion Image). E-cadherin levels were significantly different for control versus 40 mJ/cm2 (n = 3). ***, P < 0.001. Inset, representative Western blot for E-cadherin from one of three separate experiments. Equal loading of protein was verified by GAPDH staining. B, confluent PMKs were harvested at the times indicated after 40 mJ/cm2 UVR. Unirradiated PMKs were included as controls. Densitometric analysis of Western data for relative E-cadherin levels normalized to GAPDH (NIH Scion Image). E-cadherin levels were significantly different for control versus 6, 24, and 72 h (n = 3). ***, P < 0.001. Inset, representative Western blot for E-cadherin from one of three separate experiments. Equal loading of protein was verified by GAPDH staining. C, supernatants from control and UV-irradiated PMK, at the UVR doses indicated, were collected at 24 h and assessed for PGE2 content by ELISA. PGE2 levels were significantly different for control versus 40 mJ/cm2 (n = 3). **, P < 0.01.

 
PGE2 enhances E-cadherin loss in vitro. Evidence from rodent carcinogenesis models, in vitro experiments with human tumorigenic cell lines, and clinical observations in humans suggests that the nonsteroidal anti-inflammatory agent indomethacin exerts its antitumorigenic activity by suppression of cyclooxygenase and PGE2 production (28, 29). To evaluate whether endogenous PGE2 played a role in the down-regulation of E-cadherin expression in UV-irradiated PMK, we tested the effect of indomethacin on E-cadherin protein expression following UVR by Western immunoblotting. Indomethacin treatment abrogated the loss of E-cadherin in UV-irradiated cells (P < 0.01; Fig. 3A ). As expected, indomethacin also inhibited PGE2 synthesis in these cells (Fig. 3B).


Figure 3
View larger version (13K):
[in this window]
[in a new window]

  		Figure 3. PGE2 induces the down-regulation of E-cadherin. A, confluent PMKs were exposed to 40 mJ/cm2 of UVR and then cultured for 24 h in the presence or absence of indomethacin (Indo; 3 µg/mL). Unirradiated PMKs were included as controls. Densitometric analysis of Western data for relative E-cadherin levels normalized to GAPDH. E-cadherin protein levels were significantly different for control versus UV (40 mJ/cm2) and UV (40 mJ/cm2) versus UV + indomethacin (n = 3). **, P < 0.01. Inset, representative Western blot for E-cadherin from one of three separate experiments. Equal loading of protein was verified by GAPDH staining. B, supernatants from control, UV-irradiated, and indomethacin-treated UV-irradiated PMKs were collected and assessed for PGE2 content by ELISA. PGE2 levels were significantly different for control versus UV (40 mJ/cm2), control versus UV (40 mJ/cm2) + indomethacin, and UV versus UV + indomethacin (n = 3). *, P < 0.05. C, confluent PMKs were pretreated with indomethacin (3 µg/mL) for 30 min and then exposed to varying concentrations of PGE2 (10–6 to 10–10 mol/L) for 24 h in the continued presence of indomethacin. Control untreated PMKs were also included. Densitometric analysis of Western data for E-cadherin protein levels normalized to GAPDH. E-cadherin levels were significantly different for control untreated versus PGE2 (10–6 mol/L) and control untreated versus PGE2 (10-8 mol/L; n = 3). **, P < 0.01. Inset, representative Western blot of E-cadherin after exogenous application of varying doses of PGE2 (10–6 to 10–10 mol/L).

 
To test whether a link between UV-induced PGE2 production and E-cadherin down-regulation is present, we next determined if exogenous application of PGE2 to cultured PMK induced a similar down-regulation of E-cadherin–mediated cell-cell contacts. PMKs were pretreated with indomethacin, to block endogenous PGE2, and exposed to varying concentrations of exogenous PGE2. The quantity of added PGE2 was verified by ELISA (data not shown). Control and UV-irradiated cells were harvested 24 h later and analyzed for E-cadherin levels by Western immunoblotting. Results show that 10–6 and 10-8 mol/L doses of PGE2 resulted in a significant attenuation of E-cadherin protein levels (P < 0.01), whereas addition of 10–10 mol/L PGE2 resulted in E-cadherin levels similar to controls (Fig. 3C).

PGE2 induces the down-regulation of E-cadherin via the EP2 receptor subtype. PGE2 exerts its effects through interactions with at least four subtypes of PGE2 receptors (EP1, EP2, EP3, and EP4). We have previously shown that proliferation of primary human keratinocytes (PHK) is stimulated by high concentrations of PGE2 through activation of low-affinity EP2 receptors (30). Because we hypothesize that PGE2 signaling through the EP2 receptor may in part mediate the disassembly of E-cadherin cell-cell contacts to permit the hyperplasia that occurs after acute UV injury, we next determined if EP2 receptor deletion in cultured PMK attenuated this UV-induced effect. WT and EP2–/– PMKs were isolated, exposed to an acute dose of 40 mJ/cm2 of UVR, and harvested 24 h later. Our data show that EP2 deletion significantly attenuated the E-cadherin decrease produced by UVR compared with WT irradiated controls (P < 0.001; Fig. 4A ).


Figure 4
View larger version (19K):
[in this window]
[in a new window]

  		Figure 4. PGE2 down-regulates E-cadherin via the EP2 receptor subtype in vitro and in vivo. A, confluent WT and EP2–/– PMKs were exposed to an acute dose of 40 mJ/cm2 of UVR. Unirradiated WT and EP2–/– PMKs were included as controls. Densitometric analysis of Western data for relative E-cadherin levels normalized to GAPDH. E-cadherin levels were significantly different for WT control versus WT UV and WT UV versus EP2–/– UV (n = 3). ***, P < 0.001. Inset, representative Western blot of E-cadherin in WT and EP2–/– PMK after UVR. Data represent one of three separate experiments. Equal loading of protein was verified by GAPDH staining. B, confluent PMKs were pretreated with indomethacin (3 µg/mL) for 30 min and then exposed to varying concentrations of butaprost (0.01–100.0 ng/mL) for 24 h in the continued presence of indomethacin. Control untreated PMKs were also included. Densitometric analysis of Western immunoblotting for E-cadherin protein levels normalized to GAPDH. E-cadherin levels were significantly different for 1, 10, and 100 ng/mL of butaprost compared with untreated controls (n = 3). ***, P < 0.001. Inset, representative Western blot of E-cadherin after exogenous application of varying doses of butaprost (0.01–100 ng/mL) from one of three separate experiments. Equal loading of protein was verified by GAPDH staining. C, Western blot analysis of E-cadherin from epidermal curettings harvested from WT and EP2 knockout (KO) SKH-1 mice 24 and 72 h after an acute dose of UVR (180 mJ/cm2). E-cadherin levels were significantly different for WT control versus 24 h WT UV, WT control versus 72 h WT UV, 24 h WT UV versus 24 h EP2–/– UV, and 72 h WT UV versus 72 h EP2–/– UV (n = 5). ***, P < 0.001. Inset, representative Western blot of E-cadherin from WT and EP2–/– mice 24 and 72 h after UVR. Unirradiated controls from both genotypes are included.

 
To determine if the highly selective EP2 receptor agonist butaprost replicated the PGE2-mediated changes we found in E-cadherin, cultured PMKs were exposed to increasing concentrations of butaprost. The results show that butaprost suppressed E-cadherin expression in a dose-dependent fashion that peaked at 100 ng/mL (P < 0.001; Fig. 4B), a dose we have previously shown effective for stimulating PHK growth.

We have shown that EP2 receptor signaling modulates E-cadherin–mediated cell-cell contacts in PMK in vitro. To establish whether a similar mechanism occurred in vivo, we next used SKH-1 hairless mice in which the EP2 receptor was selectively deleted. In this series of experiments, WT and EP2–/– mice were exposed to an acute dose of 180 mJ/cm2 UV and sacrificed 24 and 72 h after irradiation. Western blot analysis shows that EP2 receptor deletion in epithelium rescued mice from this UV-induced loss of E-cadherin at both time points evaluated (P < 0.05; Fig. 4C).

E-cadherin down-regulation precedes cellular proliferation. Previous studies by our group clearly show that PGE2 secretion and cell proliferation after UVR are linked (11, 31, 32). To determine whether UV-PGE2–induced down-regulation of E-cadherin is coupled to proliferation, we did a more detailed time course analysis of E-cadherin, cyclin D1, and Ki67 expression by Western immunoblotting in confluent PMK after exogenous addition of PGE2. A time-dependent decrease in E-cadherin expression was observed as early as 2 h, with levels further decreasing 72 h after UV exposure (P < 0.05; Fig. 5A ). In contrast, levels of cyclin D1 declined gradually and remained relatively low for the first 24 h after addition of PGE2. By 72 h, a marked increase in cyclin D1 levels was observed (P < 0.001). Levels of Ki67, in comparison with cyclin D1, were low to undetectable 72 h after treatment. These Western blot results for Ki67 were validated by immunofluorescence microscopy6 and by control lysates prepared from SCCs resected from mice exposed to a 30-week UVR protocol. Because PGE2 stimulates proliferation by EP2 receptor activation, we next evaluated cyclin D1 and Ki67 levels in PMK treated with the EP2 receptor agonist butaprost (Fig. 5B). A similar temporal lag or uncoupling between E-cadherin loss and cell proliferation was observed by Western immunoblotting (Fig. 5B).


Figure 5
View larger version (23K):
[in this window]
[in a new window]

  		Figure 5. PGE2-EP2–mediated E-cadherin loss is uncoupled to cellular proliferation. A, confluent PMKs were pretreated with indomethacin (3 µg/mL) for 30 min and then exposed to PGE2 (10–6 mol/L) in the continued presence of indomethacin for the indicated times. Cellular protein was extracted and levels of E-cadherin and cyclin D1 were analyzed by Western blotting. E-cadherin levels were significantly different for control versus 2, 6, 24, and 72 h (n = 3). ***, P < 0.001. Cyclin D1 levels were statistically significant for control versus 72 h and 72 h WT UV versus 72 h EP2–/– UV (n = 3). ***, P < 0.001. Inset, representative Western blot of E-cadherin, cyclin D1, and Ki67 from one of three separate experiments. Control PMKs are included. Undetectable levels of Ki67 were observed in PMK 72 h after treatment with exogenous PGE2. SCCs from chronically UV-irradiated SKH-1 mice were included as controls. Equal loading of protein was verified by GAPDH. B, confluent PMKs were pretreated with indomethacin (3 µg/mL) for 30 min and then exposed to butaprost (100 ng/mL) in the continued presence of indomethacin for the indicated times. Control PMKs are included. Representative Western blot of E-cadherin, cyclin D1, and Ki67 from one of two separate experiments. Undetectable levels of Ki67 were observed in PMK 72 h after treatment with butaprost. SCCs from chronically UV-irradiated SKH-1 mice were included as controls. Equal loading of protein was verified by GAPDH. C, WT and EP2–/– mice were exposed to an acute 180 mJ/cm2 dose of UVR and sacrificed 24 and 72 h later. Skin sections were formalin fixed, paraffin embedded, and immunostained for Ki67 by immunohistochemistry. The number of Ki67-positive epidermal keratinocytes was counted in 10 randomly selected regions from each slide under 40x objective. The average number of stained cells and SD for each group (SE) were calculated. Ki67 levels were statistically significant for WT control versus 72 h WT UV, WT control versus 72 h EP2–/– UV, and 72 h WT UV versus 72 h EP2–/– UV (n = 3). ***, P < 0.001.

 
To validate that this early EP2-mediated down-regulation of E-cadherin is not temporally coupled to proliferation in vivo, we next examined keratinocyte proliferation using Ki67 immunostaining in UV-exposed WT and EP2–/– mice. Ki67 staining was minimal in the epidermis of unirradiated control and EP2–/– mice and remained unchanged in both genotypes 24 h after UV exposure (Fig. 5C). However, by 72 h after irradiation, EP2–/– mice showed significantly reduced keratinocyte proliferation compared with irradiated WT mice (P < 0.05). Taken together, these data show a temporal uncoupling between early UV-induced PGE2-EP2–mediated E-cadherin loss and cellular proliferation, suggesting that some other mechanism(s) for this down-regulation of E-cadherin–mediated cell-cell contacts is involved.

Lysosomal and proteasomal activity is required for UV-EP2–mediated E-cadherin down-regulation. Recent studies suggest that posttranscriptional processes involving E-cadherin endocytosis, endosomal sorting, and lysosomal-mediated degradation play a major role in dynamically modulating E-cadherin levels and activity at the cell surface (33–35). Therefore, using immunofluorescent microscopy, we investigated whether the subcellular distribution of E-cadherin changed after an acute UV exposure in PMK in vitro. In control PMK, E-cadherin staining was localized to lateral surfaces of the cell at sites of cell-cell contacts (Fig. 6A ). In contrast, WT UV-irradiated PMK showed a redistribution of E-cadherin staining from the plasma membrane surface into vesicular structures in the cytosol. A similar redistribution of E-cadherin staining into these intracellular vesicles was shown after exogenous addition of PGE2 or butaprost (Fig. 6B).


Figure 6
View larger version (41K):
[in this window]
[in a new window]

  		Figure 6. E-cadherin internalization and degradation in the lysosome and proteasome. A, PMKs were seeded onto collagen-1–precoated chamber slides, grown to confluency, and treated with an acute 40 mJ/cm2 dose of UVR. Twenty-four hours later, cells were fixed in 4% paraformaldehyde and labeled using polyclonal rabbit anti-E-cadherin (Santa Cruz Biotechnology). Specific binding was detected using an Alexa Fluor 488–conjugated secondary antibody (green). Hoechst counterstain (blue) was used to label nuclei. Slides were examined on an Olympus fluorescent microscope. Representative sections from three independent experiments. Images are from one of five representative fields. Control unirradiated PMKs are included. Arrows, membrane staining for E-cadherin; arrowheads, localization of E-cadherin staining in cytoplasmic vesicles. B, PMKs were seeded onto collagen-1–precoated chamber slides, grown to confluency, and treated with either 10–6 mol/L PGE2 or 100 ng/mL butaprost for 24 h. Cells were fixed in 4% paraformaldehyde and labeled using polyclonal rabbit anti-E-cadherin. Green, specific binding was detected using an Alexa Fluor 488–conjugated secondary antibody. Hoechst counterstain (blue) was used to label nuclei. Control untreated PMKs are included. Representative sections from three independent experiments. Arrows, membrane staining for E-cadherin; arrowheads, localization of E-cadherin staining in cytoplasmic vesicles. C, WT and EP2–/– PMKs were seeded onto collagen-1 six-well plates, grown to confluency, and then pretreated with the lysosome inhibitor chloroquine (chloro; 50 µmol/L) or the proteasome inhibitor ALLN (50 µmol/L; Sigma) for 1 h before UVR (40 mJ/cm2). After 24 h, cells were lysed, subjected to SDS-PAGE, transferred to nitrocellulose membranes, and analyzed by Western blotting. E-cadherin levels, normalized to GAPDH, were significantly different for WT control versus WT UV, WT UV versus UV + chloroquine, WT UV versus UV + lactacystin, and WT UV versus EP2–/– UV (n = 4). **, P < 0.01.

 
Because both lysosomes and proteasomes are important mediators in the degradation of cellular proteins, we preincubated UV-irradiated WT and EP2–/– PMK with drugs that cause inhibition of these pathways. Cells were pretreated with the lysosomal inhibitor chloroquine (50 µmol/L) or the proteasome inhibitor ALLN (50 µmol/L) for 1 h before UVR. As shown in Fig. 6C, Western immunoblotting revealed a significant decrease in the expression of E-cadherin in WT PMK following UVR, which was partially attenuated by pretreatment of the cells with chloroquine (P < 0.01) and totally prevented by ALLN (P < 0.01). In contrast, these inhibitors had no effect on E-cadherin expression levels in EP2–/– PMK after UVR. Taken together, we can conclude that UVR induces the early loss of E-cadherin expression in keratinocytes via a posttranslational mechanism involving the mobilization of this adhesion molecule from the cell surface to lysosome/proteasome proteolytic pathways for processing/degradation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
E-cadherin down-regulation in the epidermis is a naturally occurring phenomenon during wound healing when the normal architecture of the epithelia is perturbed and requires remodeling. Evidence also supports a causal role for E-cadherin loss in the development and progression of a wide array of epithelial cancers (2–6, 36). Loss of E-cadherin has been correlated in clinical studies with various pathologic and clinical features, such as tumor dedifferentiation, infiltrative growth, and a poorer patient prognosis (37, 38). In addition, UVR, a potent skin cancer inducer, has been shown to down-regulate E-cadherin expression in human tissue skin constructs in vitro; similar results have also been shown in human malignant skin lesions, suggesting that E-cadherin down-regulation plays a critical role in SCC progression (7–9).

In the present study, we used the well-established SKH-1 hairless mouse photocarcinogenesis model (22, 24, 25) to investigate the potential mechanisms by which UVR regulates E-cadherin expression in skin keratinocytes. E-cadherin changes were studied acutely, both in vitro and in vivo, as well as chronically in vivo as irradiated epithelium developed early dysplasia, which progressed over time to become invasive SCCs. In the work presented here, we show that SCC progression by UVR produces a significant and progressive down-regulation of E-cadherin protein expression in epidermal keratinocytes as lesions progress from in situ carcinomas to large invasive SCCs. We also show an early loss of E-cadherin expression in epidermal keratinocytes 24 h after a single UV exposure. Although this loss of E-cadherin is a common feature of several types of malignancies, it is unclear if this loss is initiated in the early or late stages of carcinogenesis. Using a transgenic mouse model of pancreatic ß-cell carcinogenesis, Perl et al. (3) reported that E-cadherin loss coincided with more advanced stages of disease as lesions transitioned from adenomas to invasive tumors. In human gastric cancers, E-cadherin loss occurred in both early and advanced lesions (39, 40). Based on our data in skin, UVR in vivo induces an early decrease in E-cadherin–mediated cell-cell contacts in precancerous lesions that progresses to a near complete loss when tumors form and become invasive SCCs. To further understand how an acute UV exposure regulates E-cadherin in vitro, we next studied cultured PMK from SKH-1 mice. We observed the same patterns of regulation, indicating that the acute UV-induced down-regulation of E-cadherin seen in vivo relates specifically to the epidermis and not due to potential cytokines or PGE2 released from dermal fibroblasts, vascular endothelial cells, or invading inflammatory cells.

Because loss of keratinocyte E-cadherin occurs in early precancerous dysplastic lesions in the epidermis when prostaglandin levels are quite high, we hypothesized that UV-induced PGE2 production may be linked and possibly responsible for the aforementioned E-cadherin down-regulation following UVR in our model system. To address this question, we next evaluated the levels of PGE2 in confluent epidermal cultures, where low physiologic levels of PGE2 were observed concurrent with the presence of ubiquitous E-cadherin–mediated cell-cell contacts. We then showed that application of PGE2 to confluent epidermal cultures directly down-regulated E-cadherin. In addition, in UVR in vitro experiments we showed that when UV-induced PGE2 production was blocked with indomethacin, the aforementioned UV-mediated loss of E-cadherin was rescued by this cyclooxygenase inhibitor. Second, we turned our attention to the EP2 receptor downstream of PGE2. Using the chronic photocarcinogenesis model, we have previously shown this receptor to be involved in the development of UV-induced skin tumors (22). Specifically, we reported that deletion of the EP2 receptor in SKH-1 hairless mice reduces the number of UV-induced skin tumors by 50% (22). The decreased number of tumors in EP2–/– animals suggested that UV-stimulated E-cadherin loss may be mediated by EP2 receptor signaling. In our present study, we used the highly selective EP2 receptor agonist butaprost, as well as EP2–/– keratinocyte cultures and mice, to show that the loss of E-cadherin–mediated cell-cell contacts is modulated by the EP2 receptor. Other investigators studying the downstream signaling potential of PGE2 have revealed its capacity to regulate the cell-cell adhesion glycoprotein ß-catenin (41, 42) and also recently E-cadherin (43). However, this latter study only showed effects of PGE2 on E-cadherin expression using cultured lung adenocarcinoma tumor cells that inherently are altered phenotypically. Taken together, these results show that PGE2, via EP2 receptor signaling, can initiate the loss of E-cadherin–mediated cell-cell contacts, although it does not address the actual mechanism by which this may be occurring.

A central question raised by our studies relates to the mechanism(s) by which UVR may mediate the aforementioned loss of E-cadherin–mediated cell-cell contacts. First, we considered whether the observed epidermal E-cadherin down-regulation is a direct effect of UVR or whether it is possibly a secondary effect of other UV-induced phenomena, such as keratinocyte proliferation. In other words, it is possible that the observed E-cadherin down-regulation may be an indirect result of proliferative signals generated by epithelial cells in response to UV injury or exogenous PGE2. Previous studies by our group as well as others suggest that UVR can induce PGE2 production and cell proliferation (22, 25, 31, 32). To this end, our data show the lack of correlation between keratinocyte proliferation and E-cadherin down-regulation: In both in vitro and in vivo experimental settings, E-cadherin loss preceded temporally the induction of keratinocyte proliferation by at least 48 h after UVR, PGE2, or butaprost (EP2 agonist) treatments. These results agree with previous studies done by our group that clearly show a 72- to 96-h delay in keratinocyte proliferation in mice after acute UVR (22, 25, 27). Moreover, in over 45 human patients in which biopsies were taken from skin exposed to a three minimal erythema dose of UVR, we observed no change in the proliferative marker bromodeoxyuridine until 72 h.7 Collectively, these data suggest that loss of E-cadherin may serve as an etiologic factor underlying the initiation of keratinocyte proliferation. In fact, a recent article by Liu et al. (44) showed a stimulatory role for E-cadherin in proliferative regulation of normal rat kidney or epithelial cells. However, additional studies are needed in the future to test this hypothesis in epidermal keratinocytes.

It is also noteworthy that E-cadherin down-regulation was observed as quickly as 6 h following UV exposure in vitro. This type of protein regulation cannot be explained by classic transcriptional or translational gene regulation. Therefore, using PMK cultures, we examined E-cadherin cellular localization by immunofluorescence microscopy in vitro following UVR, PGE2, or butaprost treatment. Our data show the early mobilization of E-cadherin from the cell surface into the cytoplasm following UV exposure. A similar internalization and rerouting of E-cadherin is shown into the cytoplasm after exogenous PGE2 or butaprost. Moreover, we then show a significant decrease of E-cadherin protein levels in WT PMK following UVR, which was partially attenuated by chloroquine, a lysosome inhibitor, and totally rescued by ALLN, a proteasome inhibitor. In contrast, we observed that these inhibitors had no effect on E-cadherin levels of expression in UV-exposed EP2–/– PMK. Based on these results, we conclude that UVR induces the early loss of E-cadherin–mediated cell-cell contacts in keratinocytes via the mobilization of this adhesion molecule from the cell surface to lysosome/proteasome proteolytic pathways for processing/degradation. To this end, there is an increasing body of evidence showing that E-cadherin is constitutively internalized into early endosomes and recycled back to the cell surface (45). Similar posttranslational processing of E-cadherin involving ubiquitination, endocytosis, and endosomal sorting to the lysosome and proteasome for degradation during the process of epithelial-mesenchymal transitions has been described in genetically modified epithelial cells (33–35, 46).

In conclusion, we report regulation of E-cadherin by acute exposure to UVR and a sequential loss of E-cadherin expression with UV-induced SCC progression in a mouse model of photocarcinogenesis. Moreover, we have presented data showing that UVR, via the PGE2-EP2 signaling pathway, may initiate tumorigenesis in keratinocytes by down-regulating E-cadherin–mediated cell-cell contacts through its mobilization away from the cell membrane, internalization into the cytoplasm, and shuttling through the lysosome and proteasome degradation pathways.


   Acknowledgments
 
Grant support: Emergency Department, Wilmot Cancer Research Fellowship, and NIH grants T32AR07472-18 and RO1 CA117821-06.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Drs. Freeman, Nehrke, Hinkle, and Kowalcyzk for all their help and guidance; Fatat Sleiman for histology assistance; and Dr. Meyowitz (Department of Eastman Dental) for allocation of laboratory space needed for resubmission of this manuscript.


   Footnotes
 
6 S. Brouxhon, unpublished observations. Back

7 A.P. Pentland, unpublished observations. Back

Received 11/30/06. Revised 4/30/07. Accepted 6/ 1/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. American Cancer Society. Cancer facts and figures, 2003. Atlanta (GA): American Cancer Society; 2003.
  2. Cavallaro U, Christofori G. Cell adhesion and signaling by cadherins and IG-CAMS in cancer. Nature 2004;4:118–32.
  3. Perl A, Wilgenbus P, Dahl U, Semb H, Christofori G. A causal role for E-cadherin in the transition from adenoma to carcinoma. Nature 1998;392:190–3.[CrossRef][Medline]
  4. Birchmeier W, Behrens J. Cadherin expression in carcinomas: role in the formation of cell junctions and the prevention of invasiveness. Biochim Biophys Acta 1994;1198:11–26.[Medline]
  5. Christofori G, Semb H. The role of the cell-adhesion molecule E-cadherin as a tumour-suppressor gene. Trends Biochem Sci 1999;24:73–6.[CrossRef][Medline]
  6. Vleminckx K, Vakaet L, Jr., Mareel M. Fiers W, van Roy F. Genetic manipulation of E-cadherin expression by epithelial tumor cells reveals an invasion suppressor role. Cell 1991;66:107–19.[CrossRef][Medline]
  7. Margulis A, Zhang W, Alt-Holland A, Crawford HC, Fusenig NE, Garlick JA. E-cadherin suppression accelerates squamous cell carcinoma progression in three-dimensional, human tissue constructs. Cancer Res 2005;65:1783–91.[Abstract/Free Full Text]
  8. Margulis A, Zhang W, Alt-Holland A, et al. Loss of intercellular adhesion activates a transition from low- to high-grade human squamous cell carcinoma. Int J Cancer 2006;118:821–31.[CrossRef][Medline]
  9. Fuller LC, Allen MH, Montesu M, Barker JN, Macdonald DM. Expression of E-cadherin in human epidermal non-melanoma cutaneous tumours. Br J Dermatol 1996;134:28–32.[CrossRef][Medline]
  10. Navarro P, Gomez M, Pizarro A, Gamallo C, Quintanilla M, Cano A. A role for the E-cadherin cell-cell adhesion molecule during tumor progression of mouse epidermal carcinogenesis. J Cell Biol 1991;115:517–33.[Abstract/Free Full Text]
  11. Pentland AP, Mahoney M, Jacobs SC, Holtzman MJ. Enhanced prostaglandin synthesis after ultraviolet injury is mediated by endogenous histamine stimulation. A mechanism for irradiation erythema. J Clin Invest 1990;86:566–74.[Medline]
  12. Pugh S, Thomas GA. Patients with adenomatous polyps and carcinomas have increased colonic mucosal prostaglandin E2. Gut 1994;35:675–8.[Abstract/Free Full Text]
  13. Rigas B, Goldman IS, Levine L. Altered eicosanoid levels in human colon cancer. J Lab Clin Med 1993;122:518–23.[Medline]
  14. Furstenberger G, Marks F. The role of eicosanoids in normal, hyperplastic and neoplastic skin. In: Ruzicka T, editor. Eicosanoids in the skin. Boca Raton (FL): CRC Press; 1990. p. 107–24.
  15. Mayoral R, Fernandez-Martinez A, Bosca L, Martin-Sanz P. Prostaglandin E2 promotes migration and adhesion in hepatocellular carcinoma cells. Carcinogenesis 2005;26:753–61.[Abstract/Free Full Text]
  16. Sheng H, Shao J, Washington MK, DuBois RN. Prostaglandin E2 increases growth and motility of colorectal carcinoma cells. J Biol Chem 2001;276:18075–81.[Abstract/Free Full Text]
  17. Timoshenko AV, Xu G, Chakrabarti S, Lala PK, Chakraborty C. Role of prostaglandin E2 receptors in migration of murine and human breast cancer cells. Exp Cell Res 2003;289:265–74.[CrossRef][Medline]
  18. Parnigotto PP, Conconi MT. Effects of prostaglandin E2 on growth, morphology, morphometry and keratin pattern of bovine corneal epithelial cells cultured in vitro. Ann Anat 1998;180:511–8.[Medline]
  19. Sonoshita M, Takaku K, Sasaki N, et al. Acceleration of intestinal polyposis through prostaglandin receptor EP2 in Apc({Delta}716) knockout mice. Nat Med 2001;7:1048–51.[CrossRef][Medline]
  20. Cahlin C, Gelin J, Andersson M, Lonnroth C, Lundholm K. The effects of non-selective, preferential-selective and selective COX-inhibitors on the growth of experimental and human tumors in mice related to prostanoid receptors. Int J Oncol 2005;27:913–23.[Medline]
  21. Sung YM, He G, Fischer SM. Lack of expression of the EP2 but not EP3 receptor for prostaglandin E2 results in suppression of skin tumor development. Cancer Res 2005;65:9304–11.[Abstract/Free Full Text]
  22. Brouxhon S, Konger R, VanBuskirk J, et al. Deletion of prostaglandin E2 EP2 receptor protects against ultraviolet (UV) induced carcinogenesis, but increases tumor aggressiveness. J Invest Dermatol 2007;127:439–46.[CrossRef][Medline]
  23. Sung YM, He G, Hwang DH, Fischer SM. Overexpression of the prostaglandin E2 receptor EP2 results in enhanced skin tumor development. Oncogene 2006;25:5507–16.[CrossRef][Medline]
  24. Black, HS, deGruijl FR, Forbes PD, et al. Photocarcinogenesis: an overview. J Photochem Photobiol B 1997;40:29–47.[CrossRef][Medline]
  25. Pentland AP, Scott G, VanBuskirk J, Tanck C, LaRossa G, Brouxhon S. Cyclooxygenase-1 deletion enhances apoptosis but does not protect against ultraviolet light-induced tumors. Cancer Res 2004;64:5587–91.[Abstract/Free Full Text]
  26. Brouxhon SM, Prasad AV, Joseph SA, Felten DL, Bellinger DL. Localization of corticotropin-releasing factor in primary and secondary lymphoid organs of the rat. Brain Behav Immun 1998;12:107–22.[CrossRef][Medline]
  27. Kyrkanides S, Miller JH, Federoff HJ. Systemic FIV vector administration: transduction of CNS immune cells and Purkinje neurons. Brain Res Mol Brain Res 2003;119:1–9.[Medline]
  28. Wang D, Dubois RN. Prostaglandins and cancer. Gut 2006;55:115–22.[Free Full Text]
  29. Chiu CH, McEntee MF, Whelan J. Discordant effect of aspirin and indomethacin on intestinal tumor burden in Apc(Min/+) mice. Prostaglandins Leukot Essent Fatty Acids 2000;62:269–75.[CrossRef][Medline]
  30. Konger R, Malaviya R, Pentland AP. Growth regulation of primary human keratinocytes by prostaglandin E receptor EP2 and EP3 subtypes. Biochim Biophys Acta 1998;1401:221–34.[Medline]
  31. Andley UP, Hebert JS, Morrison AR, Reddan JR, Pentland AP. Modulation of lens epithelial cell proliferation by enhanced prostaglandin synthesis after UVB exposure. Invest Ophthalmol Vis Sci 1994;35:374–81.[Abstract/Free Full Text]
  32. Regan JW. EP2 and EP4 prostanoid receptor signaling. Life Sci 2003;74:143–53.[CrossRef][Medline]
  33. Fujita Y, Krause G, Scheffner M, et al. Hakai, a c-Cbl-like protein, ubiquitinates and induces endocytosis of the E-cadherin complex. Nat Cell Biol 2002;4:222–31.[CrossRef][Medline]
  34. Palacios F, Tushir JS, Fujita Y, D'Souza-Schorey C. Lysosomal targeting of E-cadherin: a unique mechanism for the down-regulation of cell-cell adhesion during epithelial to mesenchymal transitions. Mol Cell Biol 2005;25:389–402.[Abstract/Free Full Text]
  35. Le TL, Yap AS, Stow JL. Recycling of E-cadherin: a potential mechanism for regulating cadherin dynamics. J Cell Biol 1999;146:219–32.[Abstract/Free Full Text]
  36. Wheelock MJ, Johnson KR. Cadherins as modulators of cellular phenotype. Annu Rev Cell Dev Biol 2003;19:207–35.[CrossRef][Medline]
  37. Asgeirsson KS, Jonasson JG, Tryggvadorttir L, et al. Altered expression of E-cadherin in breast cancer: patterns, mechanisms and clinical significance. Eur J Cancer 2000;36:1098–106.[CrossRef][Medline]
  38. Heimann R, Lan F, McBride R, Hellman S. Separating favourable from unfavourable prognostic markers in breast cancer: the role of E-cadherin. Cancer Res 2000;60:298–304.[Abstract/Free Full Text]
  39. Chan AO, Wong BC, Lan HY, et al. Deregulation of E-cadherin-catenin complex in precancerous lesions of gastric adenocarcinoma. J Gastroenterol Hepatol 2003;18:534–9.[CrossRef][Medline]
  40. Joo YE, Rew JS, Kim HS, Choi SH, Park CS, Kim SJ. Changes in the E-cadherin-catenin complex expression in early and advanced gastric cancers. Digestion 2001;64:111–9.[CrossRef][Medline]
  41. Castellone MD, Teramoto H, Williams BO, Druey KM, Gutkind JS. Prostaglandin E2 promotes colon cancer cell growth through a Gs-axin-ß-catenin signaling axis. Science 2005;310:1504–10.[Abstract/Free Full Text]
  42. Shao J, Jung C, Liu C, Sheng H. Prostaglandin E2 stimulates the ß-catenin/T cell factor-dependent transcription in colon cancer. J Biol Chem 2005;280:26565–72.[Abstract/Free Full Text]
  43. Dohadwala M, Yang SC, Luo J, et al. Cyclooxygenase-2-dependent regulation of E-cadherin: prostaglandin E(2) induces transcriptional repressors ZEB1 and snail in non-small cell lung cancer. Cancer Res 2006;66:5338–45.[Abstract/Free Full Text]
  44. Liu WF, Nelson CM, Pirone DM, Chen CS. E-cadherin engagement stimulates proliferation via Rac1. J Cell Biol 2006;173:431–41.[Abstract/Free Full Text]
  45. Kamei T, Matozaki T, Sakisaka T, et al. Coendocytosis of cadherin and c-Met coupled to disruption of cell-cell adhesion in MDCK cells—regulation by Rho, Rac and Rab small G proteins. Oncogene 1999;18:6776–84.[CrossRef][Medline]
  46. Janda E, Nevolo M, Lehmann K, Downward J, Beug H, Grieco M. Raf plus TGFß-dependent EMT is initiated by endocytosis and lysosomal degradation of E-cadherin. Oncogene 2006;25:7117–30.[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Brouxhon, S.
Right arrow Articles by Pentland, A. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Brouxhon, S.
Right arrow Articles by Pentland, A. P.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online