UV-induced EGFR signal transactivation is dependent on proligand shedding by activated metalloproteases in skin cancer cell lines

Authors


Abstract

Exposure to extensive ultraviolet (UV) rays is a major cause of skin cancer, which is thought to be initiated by DNA mutations. Members of the epidermal growth factor receptor (EGFR) family are important in various pathophysiologic processes like cancer and are shown to be phosphorylated upon UV exposure. Here we show that EGFR phosphorylation by modest UV doses is dependent on metalloprotease activity and resultant epidermal growth factor (EGF) family proligand shedding. This proligand cleavage releases the mature ligand, which then binds to and activates EGFR. We show that UV induced EGFR phosphorylation in transformed cell lines of melanocyte and keratinocyte origin, which was reduced upon preincubation with a broad-spectrum metalloprotease inhibitor, BB94. UV also activated EGFR downstream signaling via Erk and Akt pathways in a BB94-sensitive manner. Furthermore, using neutralizing antibodies we found that proligand amphiregulin was required for UV-induced EGFR activation in SCC-9 cells. Using RNAi this EGFR activation was further shown to depend on the metalloproteases ADAM9 and ADAM17 in SCC-9 cells. cDNA array hybridization and RT-PCR analysis showed overexpression of a Disintegrin and a Metalloproteases (ADAMs) and EGF family proligands in melanoma cell lines. Additionally, blocking EGFR signal transactivation by BB94 led to increased apoptosis in UV-irradiated cells. EGFR signal transactivation also led to increased stability of the DNA repair protein, PARP, under UV stress. Thus, both antiapoptotic and DNA repair pathways are activated simultaneously by EGFR signal transactivation. Together, our data provide novel insights into the mechanism of UV-induced EGFR activation, suggesting broad relevance of the UV-ADAM-proligand-EGFR-Erk/Akt pathway and its significance in skin cancer. © 2008 Wiley-Liss, Inc.

The epidermis is the outermost layer of skin and is comprised of keratinocytes (∼90%), melanocytes (5-10%) and occasional Merkel and Langerhans cells. Skin is also a site that is constantly exposed to sunlight that is a natural source of ultraviolet (UV) radiation. UV exposure accounts for approximately 65% of melanomas and 90% of basal and squamous cell carcinomas.1, 2 UV alone has the potential to initiate transformation and propagate the oncogenic state. The whole UV spectrum is potentially oncogenic with ultraviolet-C (UVC) being the most potent carcinogen followed by ultraviolet-B and -A (UVB and UVA).3 Cytosolic actions of UV include damage to proteins and other macromolecules, which is primarily mediated by reactive oxygen species (ROS).4 UV produces ROS as a result of photolysis of water molecules. Moreover, UV plays a central role in carcinogenesis by directly inducing DNA damage like the formation of cyclobutane pyrimidine dimers and other photoproducts and by formation of single-strand breaks and oxidation of the bases like 8-oxoG which is mediated by ROS.5, 6

Indirect activation of receptor tyrosine kinases (RTKs) by UV has been described previously.7, 8 ROS produced by UV reversibly inactivate protein tyrosine phosphatases (PTPs), which are the negative control elements of RTKs. PTP1B for example directly associates with and dephosphorylates epidermal growth factor receptor (EGFR), and therefore, acts as its negative regulator.9 ROS mediate PTP inactivation by oxidizing cysteines at their catalytic center.10 A variety of PTPs have been shown to be susceptible to inactivation by UV-induced ROS production.11, 12 UVA and UVB have additionally been shown to irreversibly inactivate PTPs via Calpain-mediated degradation showing another mechanism of UV-induced RTK activation.13

EGFR signal transactivation is another pathway shown to activate the receptor indirectly, which is thought to be a major mechanism by which G-protein-coupled receptor (GPCR) agonists' stimulation leads to EGFR activation.14 This pathway involves activation of metalloproteases belonging to A Disintegrin and A Metalloprotease (ADAM) family, which in turn cleave membrane-anchored proligands of the epidermal growth factor (EGF) family.15 The soluble ligand is then transferred to the extracellular matrix and binds to the EGFR extracellular ligand binding domain leading to receptor dimerization and activation. EGFR signal transactivation is prevalent in normal physiologic functions.16 This pathway is also active in various cancer cell types which exploit it for their survival, proliferation and motility.17, 18

Given the role of UV in skin cancer and its ability to phosphorylate EGFR, we set out to explore the possibility of EGFR signal transactivation, and its possible relevance in skin carcinogenesis. We used a UVC source in this study for its ability to induce higher EGFR phosphorylation compared to UVA/UVB.7 Various cell lines from the keratinocyte and melanocyte lineages e.g., melanoma cell lines, immortalized keratinocytes and squamous cell carcinoma cell lines were treated with modest UVC doses. We uncovered a novel mechanism of UV-induced EGFR activation in both cell types: an EGFR signal transactivation pathway under UV irradiation requiring metalloprotease-dependent proligand shedding.

Abbreviations

ADAM, A disintegrin and A metalloprotease; EGFR, epidermal growth factor receptor; GPCR, G-protein coupled receptors; pEGFR, phospho-EGFR; pY, phospho-tyrosine; ROS, reactive oxygen species; UV, ultraviolet radiation.

Material and methods

Reagents and antibodies

EGF was purchased from Sigma (Taufkirchen, Germany). Batimastat (BB94) was from British Biotech (Oxford, UK). Small interfering RNAs (siRNAs) were purchased from Dharmacon research and Ambion Inc. Anti-phospho-p44/42 MAP Kinase, anti-phospho-Akt (Ser473), and PARP antibodies were bought from Cell Signaling Technology (Beverly, MA). The antibody against human EGFR (108.1) has been characterized before.15 4G10 monoclonal antibody to detect phosphotyrosine and EGFR reblot antibodies were from Upstate Biotechnology (Lake Placid, NY). Neutralizing antibodies were purchased from R&D systems (Wiesbaden, Germany).

Cell culture

C8161 cells were kind gift from R. Gillies; HaCaT, RPMI7951, SCC-9, and other cell lines were purchased from ATCC (Rockville, MD) and propagated according to supplier's protocol. Normal melanocytes were purchased from PromoCell (Heidelberg, Germany) and propagated according to supplier's protocol. Cells were seeded at appropriate densities in 6 cm plates, and starved for 24 hr with 2 time medium change unless otherwise indicated.

Fluorescence activated cell sorting analysis

Overnight starved cells were irradiated with indicated doses of UV and harvested 20 hr postirradiation by trypsinization. Cells were collected by spinning them down at 2,000 rpm in a tabletop cooling centrifuge. Cells were resuspended in hypotonic citrate buffer (0.1% Triton X-100, 0.1% Sodium Citrate, 20 μM Propidium Iodide) and incubated in dark for 2 hr. Fluorescence activated cell sorter (FACS) analysis was done with FACScalibur (Becton Dickinson Biosciences). Sub-G0 population was counted as the apoptotic population and represented as fraction of the total cells counted.

UV treatment

Cells were exposed to UV with Stratalinker 2000 (Stratagene, La Jolla, CA) with representative wavelength of 254 nm (UVC). Cells were exposed to indicated doses of UV without lids, containing 2 ml medium in 6 cm plates, and lysed with appropriate lysis buffer after the indicated period of time post-UV exposure.

RT-PCR and primers

Primers to detect the expression of the proteins is tabulated below. RNA was isolated from the cell lines using RNeasy mini kit (Qiagen, Hilden, Germany). Equal amounts of total RNA were taken and reverse transcribed with AMV Reverse Transcriptase (Roche, Mannheim, Germany). Primers for PCR have been described elsewhere.19 PCR products were subjected to electrophoresis on a 2% agarose gel and DNA was visualized by ethidium bromide staining.

cDNA array hybridization

Total RNA, Poly(A)+ RNA and cDNA probes were generated according to the method of Chomczynski and Sacchi.20 Labeling of 3-5 μl of cDNA was performed with the Megaprime kit (Amersham, Arlington Heights, IL) in the presence of 50 μCi of [α-32P] dATP. The prehybridization solution was replaced by the hybridization solution containing 5XSSC, 0.5% (v/v) SDS, 100 Ag/ml baker's yeast tRNA (Roche) and the labeled cDNA probe (2-5 × 106 cpm/ml) and incubated at 68°C for 16 hr. Membranes were washed under stringent conditions. A phosphorimager system (Fuji BAS 1000; Fuji) was used to quantify the hybridization signals. Average values for each spot were calculated using the formula: A = (AB-B) × 100/B [A, final volume; AB, intensity of each spot signal (pixel/mm2); B, background (pixel/mm2)].

RNA interference

Transfection of 21-nucleotide siRNA duplexes (Dharmacon Research, Lafayette, CO) for targeting endogenous genes was carried out using OligofectAMINE (Invitrogen, San Diego, CA) and 10 μg siRNA duplex per 3.5 cm plate as described previously19 Transfected cells were serum starved and assayed 24 hr after transfection.

PARP cleavage assay

Nuclear protein Poly(ADP-ribose) Polymerase (PARP) was detected using antibody purchased from Upstate. Cleavage of this 116 kDa protein leads to fragment of 89 kDa and 26 kDa. 89 kDa cleavage product and the full length are recognized by the antibody. Appearance of the 89 kDa fragment was taken as an early indicator of apoptosis. Cells were lysed after indicated period of time post-UV irradiation with RIPA lysis buffer with the composition Tris-HCl: 50 mM, pH 7.4; NP-40: 1%; Na-deoxycholate: 0.25%; NaCl: 150 mM; EDTA: 1 mM; PMSF: 1 mM; aprotinin, leupeptin, pepstatin: 1 μg/ml each; sodium orthovanadate: 1 mM; sodium fluoride: 1 mM.

Cell stimulation, immunoprecipitation and immunoblotting

Stimulation of cells with UV was done with 50 J/m2 unless indicated otherwise, and lysis was done 15 minutes after irradiation with UV unless otherwise indicated. Cells were treated with EGF (5 ng/ml) for 5 minutes unless indicated otherwise. After irradiation the cells were lysed for 20 minutes on ice in buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10% glycerol, 10 mM sodium pyrophosphate, with 2 mM sodium orthovanadate, 10 mM sodium fluoride, 1 mM PMSF and 10 μg/ml aprotinin added freshly. Lysates were precleared at 13,000 rpm in a cooling tabletop centrifuge for 15 minutes, 4°C. Protein concentration was determined from supernatant aliquots using the BCA protein assay kit (Pierce, Rockford, IL), and equal amounts of protein were subjected to either gel electrophoresis or western blotting or were subjected to immunoprecipitation. Precleared lysates were immunoprecipitated using respective antibodies and 20 μl of protein A-Sepharose for 4 hr at 4oC. Treatment of the samples was then carried out as mentioned earlier.21

Results

UVC irradiation induces phosphorylation of EGFR and downstream signaling molecules in a time- and dose-dependent manner

To investigate the effect of UV irradiation on skin cells, we initially employed 2 cell lines: C8161 (secondary melanoma), and HaCaT (immortalized keratinocytes), originating from melanocytes and keratinocytes, respectively, representing the major constituent cell types of the epidermis. In C8161 cells (Fig. 1a) we found an increase in EGFR phosphorylation upon UV exposure compared to untreated control cells. pEGFR levels were detected as early as 10 minutes post-UV irradiation and remained high for up to 2 hr, returning to lower levels after 6 hr. HaCaT cells showed similar effects (Fig. 1a), where UV irradiation led to EGFR phosphorylation starting at 10 minutes post-UV irradiation, and returning to basal levels after 6 hr. The cells were then treated with increasing dose of UVC irradiation (0-500 J/m2) for 15 minutes. In a squamous cell carcinoma cell line, SCC-9 and HaCaT cells (Fig. 1b) we observed that EGFR phosphorylation increases with increasing doses of UV irradiation at lower doses of up to 100 J/m2. For doses above 100 J/m2 the increase in EGFR phosphorylation was much less prominent. So in our study 0-100 J/m2 of UV dose represents the most responsive range. Notably, a 10-20 minutes time period after UV exposure is also the most dynamic range for observing EGFR phosphorylation.

Figure 1.

UVC irradiation induces phosphorylation of EGFR in a time and dose-dependent manner. (a) C8161 and HaCaT cells were seeded at 180,000 and 400,000 cells/6 cm plate, respectively. Cells were exposed to 50J/m2 UV 24 hr after serum starvation, and lysed at the indicated time points. Lysates were immunoprecipitated (IP) for EGFR, blotted, probed for pY and reprobed for EGFR as loading control. EGF treatment (5 ng/ml, 5 minutes) was taken as positive phosphorylation control. (b) C8161 and HaCaT cells were seeded and starved as in (a), treated with indicated doses of UVC (J/m2), and lysed 15 minutes after irradiation and processed as described under (a). (c) HaCaT cells were treated as in (a), immunoblotted and probed for pAkt and pErk. Same membrane was reprobed for total Akt as loading control. (d) HaCaT cells were irradiated with indicated UV doses and lysed after 15 minutes. Immunoblotting was done for pErk, pAkt and reprobed for total Akt and Erk as loading control.

EGFR activation is linked to the activation of downstream signaling molecules, which mediate signaling pathways and the corresponding cellular processes. The activation of downstream signaling molecules Erk and Akt was observed in a UV-dependent time course. Erk and Akt phosphorylation appeared after 15 minutes with Erk phosphorylation visible up to 1 hr and Akt phosphorylation up to 4 hr in HaCaT (Fig. 1c) and C8161 (not shown) cells. In another experiment, UV led to a dose-dependent increase in phosphorylation of Erk and Akt in HaCaT cells (Fig. 1d) with their phosphorylation detectable at 20 J/m2 and 50 J/m2, respectively.

Phosphorylation of EGFR by UV can be blocked by the metalloprotease inhibitor, BB94

The metalloprotease inhibitor, BB94 is a broad-range inhibitor of ADAM subfamily of metalloproteases. Many of these proteases have been shown to be involved in EGFR signal transactivation. Upon activation by a GPCR agonist they cleave members of the EGF family of proligands releasing their active forms, which bind to and activate EGFR.15 Preincubation of C8161 and HaCaT cells with BB94 followed by UV exposure led to decreased phosphorylation of EGFR as compared to UV exposure alone (Figs. 2a and c). Solvent control, DMSO did not show any effect on UV-induced phosphorylation (Figs. 2a and c). Another metastatic melanoma cell line RPMI7951 (Fig. 2b) and SCC-9 cells (Fig. 2d) showed higher reduction of UV-induced phosphorylation of EGFR upon BB94 preincubation. These results demonstrate a metalloprotease-mediated mode of EGFR activation in these cells under our experimental conditions.

Figure 2.

Metalloprotease inhibitor (BB94) reduces phosphorylation of EGFR induced by UV irradiation. C8161 (a), RPMI7951 (b), HaCaT (c), and SCC-9 (d) cells were preincubated with BB94 (10 μM, 30 minutes) and irradiated with indicated UV doses. Cells were lysed 15 minutes postirradiation, immunoprecipitated for EGFR, blotted and probed for pY and reprobed for total EGFR levels as loading control. DMSO was taken as solvent control.

Signaling molecules downstream of EGFR show inhibition of UV-induced phosphorylation by metalloprotease inhibition

Activation of the PI3K/Akt and the MAPK1/2 pathways has been shown to be linked to activation of members of the EGFR family, either by direct interaction with the activated receptor or via adaptor molecules.22 As shown in Figures 1c and d phosphorylation of Erk and Akt in response to UV exposure was observed shortly after phosphorylation of the EGFR. We further investigated if these pathways are linked to UV-induced EGFR signal transactivation. Preincubation with BB94 reduced pErk levels in HaCaT (Fig. 3a) and SCC-9 (Fig. 3b) cells induced by UV. Reduction in pAkt levels, however, was significant only in SCC-9 cells (Fig. 3b). This experiment shows that the phosphorylation of Erk and Akt is dependent on the EGFR signal transactivation mechanism requiring metalloprotease activity.

Figure 3.

BB94 abrogates EGFR downstream signaling induced by UV. (a) Starved HaCaT and (b) SCC-9 cells were preincubated with BB94 (10 μM, 1 hr) and irradiated with 50J/m2 UV and lysed after 15 minutes. Lysates were blotted and membranes were probed for pErk and pAkt and reprobed for Akt and Erk as loading controls.

Finding the proligand responsible for the activation of EGFR under UV stress cleaved by the metalloprotease

In the GPCR-induced activation of EGFR, metalloprotease activation leads to cleavage of membrane-anchored proligands of the EGF family.15 We investigated the role of extracellular ligand binding event for EGFR activation under UV stress to show it to be a pathway involving extracellular events. Ligand binding to the extracellular domain of monomeric EGFR leads to a conformational change exposing the dimerization domain, which bridges 2 monomers forming an active dimer.23 Antibodies directed against the extracellular domain of the EGFR compete with ligand binding, interfering with the extracellular mechanism of ligand-induced EGFR activation.24 C8161 and HaCaT cells were preincubated with these blocking antibodies followed by irradiation with 50 J/m2 UV for 15 minutes. Preincubation with blocking antibodies led to a reduction in UV-induced EGFR phosphorylation in both C8161 (Fig. 4a) and HaCaT (Fig. 4b) cells, showing extracellular ligand binding necessary for EGFR activation.

Figure 4.

EGFR ligand binding domain blocking antibodies, and neutralizing antibodies against EGF family ligands inhibit UV-induced EGFR signal transactivation. C8161 (a) and HaCaT (b) cells were preincubated (1 hr) with blocking antibodies (bAbs) against EGFR ligand binding domain, and control goat antibody (cAb). Cells were then treated with 50 J/m2 and lysed after 15 minutes. Lysates were immunoprecipitated for EGFR, probed for pY, and reprobed for EGFR. (c) SCC-9 cells were pretreated for 1 hr with neutralizing antibodies against Amphiregulin (AR, 1 μg/ml), Transforming Growth Factor-alpha (TGFα, 1 μg/ml), Epiregulin (EREG, 1 μg/ml) Betacellulin (BTC, 0.6 μg/ml), and Heparin binding EGF-like growth factor (HB-EGF, 4 μg/ml). Cells were then irradiated with 60 J/m2 UV and lysed after 15 minutes to test for pY and EGFR levels after EGFR immunoprecipitation. (d) SCC-9 cells were pretreated with blocking antibodies, treated, and lysed as in (c). Equal amounts of lysates were blotted and probed for pErk and pAkt; and reprobed for Erk and Akt.

Members of the EGF ligand family are synthesized as membrane spanning precursor forms and have been shown to be cleaved by metalloproteases to produce soluble ligands.25, 26 Commercial neutralizing antibodies binding to proligands of the EGF family and interfering with their processing and binding to the EGFR are available. We preincubated SCC-9 cells with these neutralizing antibodies against individual proligands of the EGF family as reported previously,19 followed by irradiation with 60 J/m2 UV for 15 minutes. Preincubation with neutralizing antibodies against amphiregulin (AR) led to a reduction in UV-induced EGFR phosphorylation (Fig. 4c). Furthermore, phosphorylation of Erk and Akt downstream of the EGFR was also reduced upon preincubation with neutralizing antibodies against amphiregulin (Fig. 4d). These results confirm that binding of amphiregulin to the EGFR extracellular ligand binding domain is required for UV-induced EGFR activation and downstream signaling.

Finding the metalloprotease responsible for proligand shedding during UV-induced EGFR signal transactivation

Metalloproteases of the ADAM family such as ADAM 9, -10, -12, -15, and -17 have been shown to shed members of the EGF ligand family and to be thereby involved in EGFR signal transactivation.17, 27 SCC-9 cells were depleted of individual ADAMs by siRNA-mediated knockdown and knockdown efficiency was confirmed by RT-PCR analysis (Fig. 5a). Controls were treated with transfection reagents only and Gl2 (siRNA against firefly luciferase gene) was used as specificity control. Depletion of ADAM9 and ADAM17 resulted in a decrease in EGFR phosphorylation induced by UV (Fig. 5b). ADAM9/17 knockdown also led to a reduction of UV-induced Erk phospohorylation (Fig. 5c). Reduction in pAkt levels, however, was significant only in ADAM17 knockdown as compared to control and siGl2-treated cells (Fig. 5c). Although ADAM15 knockdown also showed decrease in pEGFR levels, but these results were not consistent and downstream signaling remained unperturbed. These results show ADAM9/17 activity to be necessary for proligand shedding under UV irradiation for activation of EGFR and downstream signaling.

Figure 5.

Effect of ADAM knockdown on EGFR signal transactivation upon UV irradiation. (a) SCC-9 cells were seeded and transfected with siRNA oligonucleotides against ADAM9, 10, 12, 15, and 17. Cells were harvested for total RNA 48 hr post-transfection and RT-PCR analysis was performed for the individual ADAMs indicated. (b) SCC-9 cells were seeded and transfected with siRNA oligonucleotides against Gl2, ADAM9, -10, -12, -15, and -17. Cells were starved 24 hr post-transfection and irradiated with 100 J/m2 UV after 24 hr of starvation and lysed 15 minutes after irradiation. Lysates were probed for pY and reprobed for EGFR. (c) SCC-9 were transfected with siRNAs, treated, and lysed as in (a). Total lysates were probed for pErk and pAkt; and reprobed for Erk and Akt.

EGFR signal transactivation by UV irradiation confers antiapoptotic advantage to the cells under UV stress

As reported earlier cancer cells frequently exploit the EGFR signal transactivation pathway to maintain important properties like proliferation, invasion, migration and antiapoptosis.17 We tried to find out whether EGFR signal transactivation could provide any biological advantage to cells under UV stress. Surprisingly, there was no significant difference in the proliferation of UV-treated cells compared to UV-treated cells preincubated with BB94 (data not shown). We then analyzed a possible antiapoptotic advantage mediated by EGFR signal transactivation. C8161 and HaCaT cells were treated with 25 and 50 J/m2 UV, respectively, in the presence or absence of BB94 for 20 hr, and analyzed for nuclear fragmentation by Flow cytometry. Our results show that metalloprotease inhibition did not significantly change the sub-G0 nuclear population of unirradiated C8161 (Fig. 6a) and HaCaT cells (Fig. 6b). UV irradiation initiated apoptosis in C8161 and HaCaT cells as observed by an increase in fraction of sub-G0 population (Figs. 6cf). However, upon preincubation with BB94, UV irradiation further increased the number of apoptotic cells in both C8161 (Figs. 6c and e) and HaCaT cells (Figs. 6d and f). The increase in apoptosis rates was 10 and 20% in C8161 (Fig. 6e) and HaCaT (Fig. 6f) cells, respectively. This experiment shows that UV-induced EGFR signal transactivation allows cells to survive for longer periods of time under UV stress, minimizing the apoptotic response to DNA damage induced by UV, and allowing mutant cells to accumulate.

Figure 6.

EGFR signal transactivation leads to protection from apoptosis under UV stress. Starved C8161 (a) (120,000/6 cm plate) and HaCaT (b) (300,000/6 cm plate) cells were incubated with BB94 (10 μM) for 20 hr and analyzed for nuclear DNA content by Flow Cytometry. Starved C8161 (c) and HaCaT (d) cells were irradiated with 25 and 50 J/m2 UV respectively after preincubation with BB94 (30′, 10 μM) and analyzed 20 hr after irradiation for nuclear DNA content by flow cytometry. Fraction of apoptotic nuclei in C8161 (e) and HaCaT (f) after irradiation with UV. Values plotted as mean ± S.D. (triplicates).

Effect of UV-induced EGFR signal transactivation on the stability of the DNA repair enzyme PARP

UV can induce apoptosis in cells by causing irreparable DNA damage. PARP, is a nuclear enzyme which is cleaved into an inactive form by caspases during apoptosis.28 PARP is also involved in DNA repair processes in single-strand break repair (SSB), base excision repair (BER) and nucleotide excision repair (NER).29–31 The cleavage of PARP was assessed upon UV irradiation. We could show that UV induced cleavage of PARP at various time points starting at 12 hr in C8161 (Fig. 7a) and at 6 hr in HaCaT cells (Fig. 7b) indicating PARP inactivation and initiation of apoptosis. Cleavage of PARP upon UV irradiation could be further increased upon BB94 preincubation in both C8161 (Fig. 7a) and HaCaT (Fig. 7b) cells at most of the times points observed. These experiments indicate that UV-induced EGFR signal transactivation allows higher concentration of active PARP molecules to be maintained in the nucleus, thus, prolonging PARP-mediated repair of potentially lethal DNA lesions.

Figure 7.

EGFR signal transactivation leads to increased stability of Nucleotide Excision Repair protein PARP under UV stress. Starved C8161 (a) and HaCaT (b) cells were irradiated with 50 J/m2 UV with or without preincubation with BB94 and lysed in RIPA buffer at indicated time points. Equal amounts of protein were blotted and probed for PARP and Tubulin as loading control.

Reactive oxygen species are involved in UV-induced EGFR signal transactivation

To find out if ROS were produced by UV photosensitizing medium components, we replaced DMEM of SCC-9 cells with warm PBS immediately before irradiation. We noticed a decrease in pEGFR levels in UV-irradiated PBS-covered cells as compared to the cells covered with DMEM (Fig. 8a). This experiment shows that UV generates ROS in DMEM which could subsequently lead to EGFR phosphorylation. In another experiment, we tried finding involvement of intracellular sources of ROS generation, one of the prominent candidates are NADPH oxidases (Nox) which produce ROS in a regulated fashion. We preincubated cells with a Nox inhibitor, DPI before UV irradiation of SCC-9 cells. DPI preincubation decreased UV-induced EGFR phosphorylation and phosphorylation of downstream molecules namely Erk and Akt in a dose-dependent manner (Figs. 8b and c). Combined together these experiments show the involvement of both intracellular and extracellular ROS in UV-induced EGFR signal transactivation.

Figure 8.

Role of ROS in UV-induced EGFR signal transactivation. (a) Starved SCC-9 cells were irradiated with indicated UV doses layered with PBS or DMEM. Cells were lysed after 15 minutes to test for pY and EGFR levels after EGFR immunoprecipitation. (b) Starved HaCaT cells were incubated with varying concentrations of DPI (0, 10, 20 μM) for 30 minutes and then irradiated with indicated doses of UV (0, 50, 100 J/m2). Cells were lysed 15 minutes postirradiation. Lysates were blotted and probed for pY and reprobed for EGFR. Increasing concentration of DPI preincubation could inhibit phosphorylation of EGFR induced by UV. (c) Starved HaCaT cells were treated as in (a) and lysed. Equal amounts of lysates were blotted and probed for pErk and pAkt; and reprobed for Erk and Akt.

Members of the EGFR signal transactivation pathway are overexpressed in melamoma cell lines

After establishing that the EGFR signal transactivation pathway provides a survival advantage to cancer cells under UV irradiation, we examined the expression of genes involved in this pathway in melanoma cell lines. cDNA array analysis of 20 melanoma cell lines showing moderate EGFR expression was performed to detect mRNA expression levels of ADAMs and proligands of the EGF family. The “overexpressing cell lines” show over a 5-fold increase in expression levels of a particular gene as compared to normal melanocytes. At least 50% of the melanoma cell lines were found to overexpress genes like ADAM9, -10, -12, -15, -17, TGFα and HB-EGF. Overexpression of amphiregulin was observed in 7 cell lines (Table I). Alternatively, mRNA overexpression of ADAM9, ADAM10 and amphiregulin was also confirmed by RT-PCR analysis. These experiments also exhibited ADAM9 overexpression in a majority of melanoma cell lines, and amphiregulin in 7 melanoma cell lines as compared to normal melanocytes (Fig. 9a). These results indicate that a variety of melanoma cell lines have the potential to exploit EGFR signal transactivation pathway for their survival advantage under UV irradiation.

Figure 9.

Relevance of EGFR signal transactivation pathway in skin cancer cell lines. (a) cDNA from 19 melanoma cell lines and normal primary melanocytes was prepared and subjected to PCR for ADAM9, ADAM10 and amphiregulin to check for relative expression levels. Tubulin is taken as loading control. (b) Scheme of UV-induced EGFR signal transactivation: UV leads to activation of ADAM9 or ADAM17 which in turn cleave proamphiregulin. Amphiregulin then diffuses and binds to EGFR leading to its activation and downstream signaling, which, then confers anti-apoptotic advantage to skin cancer cells via downstream signaling molecules like Erk and Akt.

Table I. Expression of ADAMs and Proligands in Melanoma Cell Lines (cDNA Array Hybridization Analysis)
GeneAccession numberNumber of cell lines showing overexpression1
  • cDNA preparation, array hybridization, and subsequent quantification was done as in materials and methods. Table shows individual genes overexpressed in the 20 melanoma cell lines showing EGFR expression.

  • 1

    Overexpression of a gene is defined as at least 5-fold higher expression compared to normal melanocytes.

ADAM 9NM_00381617
ADAM 10NM_00111010
ADAM 12NM_00347411
ADAM 15NM_20719117
ADAM 17NM_00318317
AmphiregulinNM_0016577
TGFαNM_00323613
HB-EGFNM_00194515
BetacellulinNM_0017293
EGFNM_0019635

Discussion

Incidence of skin cancer is on a rise and much of it can be attributed to exposure to UV radiations from sunlight.32 Members of the EGFR family have been shown to be overexpressed in skin cancer.33 In this study, we show a novel mechanism of EGFR activation upon UVC irradiation, using various cell lines derived from cancerous skin cells i.e., the cells mostly exposed to UV radiations from sunlight (Fig. 9b). In these cell lines, UV-induced EGFR activation is dependent on metalloprotease activity and proligand shedding, a pathway mechanistically similar to EGFR signal transactivation. Furthermore, at low doses of UVC used in the study we found this pathway to be the predominant mode of EGFR activation. Our results further our understanding of the role of UV in intracellular signal transmission and its relevance in cancer in addition to its nuclear effects. More importantly, the biological significance of this pathway can not be overemphasized by our findings that skin cancer cells exploit this pathway to survive longer under UV irradiation, and repair potentially lethal DNA lesions.

EGFR signal transactivation involves metalloprotease activity and shedding of proligands of the EGF family.14, 15, 34 We describe how a similar mechanism of UVC-induced EGFR activation dependent on metalloprotease activity and proligand shedding in skin cancer cell lines. EGFR activation by UV has been described before, where ROS produced by UV can inactivate PTPs, which are negative regulators of EGFR phosphorylation.7 However, at low doses of UVC in our studies we observed some contrasting features of EGFR activation. One of the first differences we observed was a delayed onset of the pEGFR signal in our study as compared with fast activation shown in the previous study (10 minutes compared to 1-5 minutes) (Fig. 1a). Second, in our study UVC induced higher EGFR phosphorylation in a dose-dependent manner but only at low doses and soon reached a plateau at around 300 J/m2 (Fig. 1b). This effect was not observed previously, where UV continued to increase pEGFR levels over a range of 50-5000 J/m2.7 At lower UV doses, however, our findings show that EGFR activation depends more on the transactivation pathway, as the BB94 inhibitor alone prevents the phosphorylation of EGFR in HaCaT (Fig. 2c) and RPMI7951 (Fig. 2b) cells completely, and to a lesser extent in C8161 (Fig. 2a) and SCC-9 (Fig. 2d) cells. Combined these results emphasize the EGFR signal transactivation pathway to be the prominent mode of EGFR activation under modest doses of UV irradiation in skin cancer cell lines. However, in some cell lines like C8161 (Fig. 2a) and SCC-9 (Fig. 2d) cells, where UV-induced EGFR phosphorylation was not completely inhibited by BB94, other mechanisms like PTP inactivation may also be involved.11

Upon observing the kinetics of activation of EGFR upon UV irradiation in both the cell lines, we found that EGFR phosphorylation appears after about 10 minutes, which is considerably longer as compared to direct EGF treatment (1 minutes) or GPCR agonist administration (3-5 minutes).15 UV leads to photolysis of water producing ROS; these ROS then mediate the biological effects that are associated with UV exposure. Delay in EGFR phosphorylation, thus, could be because of delay in ROS generation. Interestingly, ROS involvement has also been shown previously in EGFR signal transactivation.21, 34 Moreover, combined with previous reports our findings expand the role of EGFR signal transactivation in situations like oxidative and osmotic stress and UV irradiation.34 Interestingly, we observed an apparent increase in molecular weight of EGFR upon UV irradiation. This decreased mobility might be the result of yet unknown posttranslational modifications, which, while not the emphasis of this study might give further insights into UV/EGFR signaling.

We found out that EGFR activation upon UV irradiation is dependent on an extracellular ligand binding event as UV-induced EGFR phosphorylation could be blocked by antibodies masking the extracellular ligand binding domain (Figs. 4a and b). Neutralizing antibodies against AR reduced UV-induced EGFR phosphorylation, showing it to be the proligand required for EGFR activation by UV (Fig. 4c). Complete inhibition by anti-AR neutralizing antibodies, however, was not observed owing probably to limitations in its potency, access, or redundancy among other EGF family members as seen in a previous report.19 Interestingly, AR is an autocrine keratinocyte growth factor, and aberrant activation or overexpression results in rapidly growing keratinocytic tumors and melanoma.35–37 AR and other EGF family members are also important in EGFR signal transactivation pathways as they are the connecting link between metalloprotease activation and EGFR activation.17, 19 We also report overexpression of members of EGF ligand family in EGFR expressing melanoma cell lines (Table I, Fig. 9a). Thus, overexpression of EGF family members in melanoma and given their role in UV-induced EGFR signal transactivation identifies them as key signaling component in skin cancer.

Most of the extranuclear effects of UV irradiation are mediated by induction of ROS generation. Given the short time scale required for the activation of the pathway (around 10 minutes) a nuclear involvement is ruled out at least in the initial stages. Looking for source of ROS generation we found that a fraction of ROS was coming from the photosensitization of media components after UV absorption (Fig. 8a). In a physiologic setting the effect of extracellularly generated ROS could be minimal. More interestingly, however, we found out that interference with ROS signaling by a chemical inhibitor of enzymes responsible for ROS generation (Nox), led to a reduction in UV-induced EGFR phosphorylation and downstream signaling (Figs. 8b and c).

ADAMs are synthesized as inactive precursors which are autoinhibited by their prodomain. ROS-mediated removal of the prodomain leading to ADAM activation has been shown previously and may well lead to EGFR activation by UV in our case.38 Additionally, both ADAMs and Nox enzymes are restricted to the same subcellular region, the cell membrane; thus, the possibility of cross-talk between these 2 pathways may be an exciting area of investigation, and possibly of therapeutic importance. Additionally, we show ADAM9, 10, 12, 15 and 17 to be overexpressed in various melanoma cell lines expressing EGFR as compared to normal melanocytes (Table I, Fig. 9a). Biochemically, we show ADAM9 and ADAM17 activity to be responsible for the EGFR activation and downstream signaling upon UV irradiation (Figs. 6a and b). ADAM9 is also shown to be expressed in melanoma within the invasive front and in various melanoma cell lines.39 Altogether, overexpression and activation of ADAMs may allow efficient utilization of the UV-induced EGFR signal transactivation pathway by skin cancer cells. Previous mechanisms of UV-induced EGFR activation show to be acting via inactivating the negative regulators of EGFR pathway, i.e., inactivation of PTPs via ROS. The pathway uncovered in this report, which is also partially ROS dependent (Fig. 8) shows a rather novel mechanism of positively enhancing EGFR pathway by increased ligand processing and metalloprotease activation.

Preincubation with a metalloprotease inhibitor led to increased sensitivity of C8161 (Fig. 6a) and HaCaT (Fig. 6b) cells to UV-induced apoptosis. EGFR signal transactivation led to activation of the PI3K/Akt pathway (Fig. 3b), which has been shown to inactivate several proteins in the pro-apoptotic pathway, like caspase9, Ask1 (Apoptosis signal-regulating kinase 1) and Bax-subfamily members like Bad leading to prolonged survival.40 Thus, the activation of PI3K/Akt pathway by EGFR signal transactivation could allow the cells to survive longer under UV stress.

Prolonged exposure to UV leads to highly damaged DNA, which if left unrepaired would direct the cells to apoptosis. But if UV-induced lethality can be overcome by repairing potentially lethal DNA lesions, cells may become predisposed to accumulating oncogenic mutations. PARP cleavage has been widely used as an early indicator of apoptosis.41 Thus, our results of PARP cleavage assay (Fig. 7) also confirm our apoptosis assay results (Fig. 6). However, numerous studies show PARP to be a key antiapoptosis mediator owing to its DNA damage repair activity, rather than just an innocent bystander cleaved by caspases.28 EGFR signal transactivation leads to prolonged stability of PARP, prolonging the NER to remove DNA lesions directly induced by UV; and single SSB and BER to remove lesions induced by ROS produced upon UV irradiation.31, 42 UV-induced EGFR signal transactivation also activated MAPK1/2 which have been shown to increase expression of DNA repair proteins XRCC1 and ERCC1.43 Thus, EGFR signal transactivation provides a mechanism for cells to survive under UV stress while accumulating mutations by sending them antiapoptotic signals, and at the same time prolonging and activating DNA repair processes to remove potentially lethal lesions induced by UV and ROS.

In the end, EGFR signal transactivation under UV irradiation holds therapeutic potential in malignancies of skin origin. Upon blockage of this pathway transformed cells may undergo apoptosis during UV exposure, thereby counteracting oncogenic processes. Blockage of EGFR signal transactivation could therefore reduce the chances of accumulating mutations that may lead to cancer progression.

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