UV-induced EGFR signal transactivation is dependent on proligand shedding by activated metalloproteases in skin cancer cell lines Bhuminder Singh, Matthias Schneider, Pjotr Knyazev and Axel Ullrich * Department of Molecular Biology, Max-Planck-Institute of Biochemistry, Martinsried, Germany 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 depend- ent 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 down- stream 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 Metallopro- teases (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 sig- nal 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. Key words: EGFR signal transactivation; UV; EGFR; metallo- protease; apoptosis 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 mela- nomas 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 fol- lowed by ultraviolet-B and -A (UVB and UVA). 3 Cytosolic actions of UV include damage to proteins and other macromole- cules, which is primarily mediated by reactive oxygen species (ROS). 4 UV produces ROS as a result of photolysis of water mole- cules. 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 re- ceptor (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 acti- vate the receptor indirectly, which is thought to be a major mecha- nism by which G-protein-coupled receptor (GPCR) agonists’ stim- ulation leads to EGFR activation. 14 This pathway involves activa- tion of metalloproteases belonging to A Disintegrin and A Metalloprotease (ADAM) family, which in turn cleave mem- brane-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 phospho- rylate 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., mela- noma 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. Material and methods Reagents and antibodies EGF was purchased from Sigma (Taufkirchen, Germany). Bati- mastat (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 (Rock- ville, MD) and propagated according to supplier’s protocol. Nor- Bhuminder Singh’s current address is: Vanderbilt University Medical Center, Nashville, TN 37232. *Correspondence to: Department of Molecular Biology, Max-Planck- Institute of Biochemistry, AmKlopferspitz 18, Martinsried, D-82152, Germany. Fax: 149-89-85782454. E-mail: ullrich@biochem.mpg.de Received 27 February 2008; Accepted after revision 19 August 2008 DOI 10.1002/ijc.23974 Published online 2 September 2008 in Wiley InterScience (www.interscience. wiley.com). 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. Int. J. Cancer: 124, 531–539 (2009) ' 2008 Wiley-Liss, Inc. Publication of the International Union Against Cancer