Ultraviolet Irradiation Can Induce Evasion of Colon Cancer Cells from Stimulation of Epidermal Growth Factor
2011; Elsevier BV; Volume: 286; Issue: 29 Linguagem: Inglês
10.1074/jbc.m111.240630
ISSN1083-351X
AutoresSeiji Adachi, Ichiro Yasuda, Masanori Nakashima, Takahiro Yamauchi, Junji Kawaguchi, Masahito Shimizu, Masahiko Itani, Momoko Nakamura, Yumi Nishii, Takashi Yoshioka, Yoshinobu Hirose, Yukio Okano, Hisataka Moriwaki, Osamu Kozawa,
Tópico(s)Cancer-related Molecular Pathways
ResumoReceptor down-regulation is the most prominent regulatory system of EGF receptor (EGFR) signal attenuation and a critical target for therapy against colon cancer, which is highly dependent on the function of the EGFR. In this study, we investigated the effect of ultraviolet-C (UV-C) on down-regulation of EGFR in human colon cancer cells (SW480, HT29, and DLD-1). UV-C caused inhibition of cell survival and proliferation, concurrently inducing the decrease in cell surface EGFR and subsequently its degradation. UV-C, as well as EGFR kinase inhibitors, decreased the expression level of cyclin D1 and the phosphorylated level of retinoblastoma, indicating that EGFR down-regulation is correlated to cell cycle arrest. Although UV-C caused a marked phosphorylation of EGFR at Ser-1046/1047, UV-C also induced activation of p38 MAPK, a stress-inducible kinase believed to negatively regulate tumorigenesis, and the inhibition of p38 MAPK canceled EGFR phosphorylation at Ser-1046/1047, as well as subsequent internalization and degradation, suggesting that p38 MAPK mediates EGFR down-regulation by UV-C. In addition, phosphorylation of p38 MAPK induced by UV-C was mediated through transforming growth factor-β-activated kinase-1. Moreover, pretreatment of the cells with UV-C suppressed EGF-induced phosphorylation of EGFR at tyrosine residues in addition to cell survival signal, Akt. Together, these results suggest that UV-C irradiation induces the removal of EGFRs from the cell surface that can protect colon cancer cells from oncogenic stimulation of EGF, resulting in cell cycle arrest. Hence, UV-C might be applied for clinical strategy against human colon cancers. Receptor down-regulation is the most prominent regulatory system of EGF receptor (EGFR) signal attenuation and a critical target for therapy against colon cancer, which is highly dependent on the function of the EGFR. In this study, we investigated the effect of ultraviolet-C (UV-C) on down-regulation of EGFR in human colon cancer cells (SW480, HT29, and DLD-1). UV-C caused inhibition of cell survival and proliferation, concurrently inducing the decrease in cell surface EGFR and subsequently its degradation. UV-C, as well as EGFR kinase inhibitors, decreased the expression level of cyclin D1 and the phosphorylated level of retinoblastoma, indicating that EGFR down-regulation is correlated to cell cycle arrest. Although UV-C caused a marked phosphorylation of EGFR at Ser-1046/1047, UV-C also induced activation of p38 MAPK, a stress-inducible kinase believed to negatively regulate tumorigenesis, and the inhibition of p38 MAPK canceled EGFR phosphorylation at Ser-1046/1047, as well as subsequent internalization and degradation, suggesting that p38 MAPK mediates EGFR down-regulation by UV-C. In addition, phosphorylation of p38 MAPK induced by UV-C was mediated through transforming growth factor-β-activated kinase-1. Moreover, pretreatment of the cells with UV-C suppressed EGF-induced phosphorylation of EGFR at tyrosine residues in addition to cell survival signal, Akt. Together, these results suggest that UV-C irradiation induces the removal of EGFRs from the cell surface that can protect colon cancer cells from oncogenic stimulation of EGF, resulting in cell cycle arrest. Hence, UV-C might be applied for clinical strategy against human colon cancers. IntroductionMembers of the EGF receptor (EGFR) 2The abbreviations used are: EGFREGF receptorUV-Cultraviolet-CMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideRbretinoblastoma proteinTAK-1TGF-β-activated kinase-1JJ/m2BrdUbromodeoxyuridineMKKMAPK kinaseSer-1046/7Ser-1046/1047. family, which are frequently overexpressed in several types of human cancers, including cancers of the lung (1Rusch V. Klimstra D. Venkatraman E. Pisters P.W. Langenfeld J. Dmitrovsky E. Clin. Cancer Res. 1997; 3: 515-522PubMed Google Scholar), head and neck (2Masuda M. Suzui M. Weinstein I.B. Clin. Cancer Res. 2001; 7: 4220-4229PubMed Google Scholar), prostate (3Fong C.J. Sherwood E.R. Mendelsohn J. Lee C. Kozlowski J.M. Cancer Res. 1992; 52: 5887-5892PubMed Google Scholar), breast (4Pianetti S. Guo S. Kavanagh K.T. Sonenshein G.E. Cancer Res. 2002; 62: 652-655PubMed Google Scholar), pancreas (5Feng F.Y. Varambally S. Tomlins S.A. Chun P.Y. Lopez C.A. Li X. Davis M.A. Chinnaiyan A.M. Lawrence T.S. Nyati M.K. Oncogene. 2007; 26: 3431-3439Crossref PubMed Scopus (62) Google Scholar), and colon (6Shimizu M. Deguchi A. Lim J.T. Moriwaki H. Kopelovich L. Weinstein I.B. Clin. Cancer Res. 2005; 11: 2735-2746Crossref PubMed Scopus (298) Google Scholar), have been associated with abnormal growth of these tumors. It is well known that exposure of cells to EGF results in rapid autophosphorylation of EGFR molecules at the cell surface (7Meisenhelder J. Suh P.G. Rhee S.G. Hunter T. Cell. 1989; 57: 1109-1122Abstract Full Text PDF PubMed Scopus (601) Google Scholar, 8Lowenstein E.J. Daly R.J. Batzer A.G. Li W. Margolis B. Lammers R. Ullrich A. Skolnik E.Y. Bar-Sagi D. Schlessinger J. Cell. 1992; 70: 431-442Abstract Full Text PDF PubMed Scopus (1331) Google Scholar, 9Rozakis-Adcock M. McGlade J. Mbamalu G. Pelicci G. Daly R. Li W. Batzer A. Thomas S. Brugge J. Pelicci P.G. Schlessinger J. Pawson T. Nature. 1992; 360: 689-692Crossref PubMed Scopus (825) Google Scholar, 10Levkowitz G. Waterman H. Ettenberg S.A. Katz M. Tsygankov A.Y. Alroy I. Lavi S. Iwai K. Reiss Y. Ciechanover A. Lipkowitz S. Yarden Y. Mol. Cell. 1999; 4: 1029-1040Abstract Full Text Full Text PDF PubMed Scopus (829) Google Scholar), which upon activation lead to cell proliferation, motility, and enhanced survival (11Zandi R. Larsen A.B. Andersen P. Stockhausen M.T. Poulsen H.S. Cell Signal. 2007; 19: 2013-2023Crossref PubMed Scopus (229) Google Scholar). There are several mechanisms by which EGFR becomes oncogenic including: 1) increased EGFR expression levels, 2) autocrine and/or paracrine growth factor loops, 3) heterodimerization with other EGFR family members and cross-talk with heterologous receptor systems, 4) defective receptor down-regulation, and 5) activating mutations (12Arteaga C.L. Oncologist. 2002; 7: 31-39Crossref PubMed Scopus (401) Google Scholar). In clinical trials, increasing evidence shows the efficacy of EGFR-targeted agents, including monoclonal antibodies on the one hand and tyrosine kinase inhibitors on the other (13Milano G. Spano J.P. Leyland-Jones B. Br. J. Cancer. 2008; 99: 1-5Crossref PubMed Scopus (41) Google Scholar).Following activation, the ligand-receptor complexes are internalized and then enter endosomes, where the receptors and their ligands are sorted to various intracellular destinations (14Massie C. Mills I.G. Nat. Rev. Cancer. 2006; 6: 403-409Crossref PubMed Scopus (65) Google Scholar). Thus, some receptors can be recycled back to the cell surface via early endosomes, and others are targeted to late endosomes and lysosomes for proteolytic degradation. There is increasing evidence that not only does receptor internalization act to terminate signaling, but that internalized endosome-associated receptors are also able to stimulate specific signal transduction pathways (15Wiley H.S. Exp. Cell Res. 2003; 284: 78-88Crossref PubMed Scopus (299) Google Scholar, 16Di Fiore P.P. De Camilli P. Cell. 2001; 106: 1-4Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, 17McPherson P.S. Kay B.K. Hussain N.K. Traffic. 2001; 2: 375-384Crossref PubMed Scopus (184) Google Scholar). Some agents that induce ligand-independent internalization and degradation of EGFR, such as the 225 mouse antibody (18Jaramillo M.L. Leon Z. Grothe S. Paul-Roc B. Abulrob A. O'Connor McCourt M. Exp. Cell Res. 2006; 312: 2778-2790Crossref PubMed Scopus (75) Google Scholar) and gemcitabine (5Feng F.Y. Varambally S. Tomlins S.A. Chun P.Y. Lopez C.A. Li X. Davis M.A. Chinnaiyan A.M. Lawrence T.S. Nyati M.K. Oncogene. 2007; 26: 3431-3439Crossref PubMed Scopus (62) Google Scholar), could have promising potential for cancer therapies. By contrast, it has previously been reported that the other factors or agents, such as oxidative stress (19Khan E.M. Heidinger J.M. Levy M. Lisanti M.P. Ravid T. Goldkorn T. J. Biol. Chem. 2006; 281: 14486-14493Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) and cisplatin (20Winograd-Katz S.E. Levitzki A. Oncogene. 2006; 25: 7381-7390Crossref PubMed Scopus (150) Google Scholar), can induce internalization of the EGFR but not degradation. They differ in their effects on the fate of the receptors, downstream signaling, and cell proliferation.Receptor down-regulation is the most prominent regulatory system of EGFR signal attenuation and involves the internalization and subsequent degradation of the activated receptor in the lysosomes. With the current knowledge of the mechanism underlying EGFR down-regulation, this molecular event involves several important phosphorylation sites in EGFR. One is the phosphorylation at Tyr-1045, which provides a docking site for the ubiquitin ligase c-Cbl resulting in ubiquitination of the EGFR (10Levkowitz G. Waterman H. Ettenberg S.A. Katz M. Tsygankov A.Y. Alroy I. Lavi S. Iwai K. Reiss Y. Ciechanover A. Lipkowitz S. Yarden Y. Mol. Cell. 1999; 4: 1029-1040Abstract Full Text Full Text PDF PubMed Scopus (829) Google Scholar). The others are the phosphorylation at serine or threonine residues that is thought to represent a mechanism for attenuation of the receptor tyrosine kinase activity (21Countaway J.L. McQuilkin P. Gironès N. Davis R.J. J. Biol. Chem. 1990; 265: 3407-3416Abstract Full Text PDF PubMed Google Scholar, 22Countaway J.L. Nairn A.C. Davis R.J. J. Biol. Chem. 1992; 267: 1129-1140Abstract Full Text PDF PubMed Google Scholar, 23Theroux S.J. Latour D.A. Stanley K. Raden D.L. Davis R.J. J. Biol. Chem. 1992; 267: 16620-16626Abstract Full Text PDF PubMed Google Scholar). Among the major sites of serine and threonine phosphorylation of the EGFR, it has previously been shown that the Ser-1046/7 phosphorylation site is required for EGFR desensitization in EGF-treated cells (23Theroux S.J. Latour D.A. Stanley K. Raden D.L. Davis R.J. J. Biol. Chem. 1992; 267: 16620-16626Abstract Full Text PDF PubMed Google Scholar). Moreover, mutant EGFR at Ser-1046/7 reportedly causes the inhibition of the EGF-induced endocytosis and down-regulation of cell surface receptors (22Countaway J.L. Nairn A.C. Davis R.J. J. Biol. Chem. 1992; 267: 1129-1140Abstract Full Text PDF PubMed Google Scholar).We have recently reported that phosphorylation of EGFR at serine 1046/7 via activation of p38 MAPK plays a pivotal role in down-regulation of EGFR induced by (−)-epigallocatechin gallate (24Adachi S. Shimizu M. Shirakami Y. Yamauchi J. Natsume H. Matsushima-Nishiwaki R. To S. Weinstein I.B. Moriwaki H. Kozawa O. Carcinogenesis. 2009; 30: 1544-1552Crossref PubMed Scopus (85) Google Scholar), anisomycin (25Adachi S. Natsume H. Yamauchi J. Matsushima-Nishiwaki R. Joe A.K. Moriwaki H. Kozawa O. Cancer Lett. 2009; 277: 108-113Crossref PubMed Scopus (30) Google Scholar), and HSP90 inhibitor (26Adachi S. Yasuda I. Nakashima M. Yamauchi T. Yamauchi J. Natsume H. Moriwaki H. Kozawa O. Oncol. Rep. 2010; 23: 1709-1714Crossref PubMed Scopus (18) Google Scholar). Moreover, there is an evidence that cisplatin also induces EGFR internalization, which is mediated by p38 MAPK-dependent phosphorylation of the receptor at threonine 669 (20Winograd-Katz S.E. Levitzki A. Oncogene. 2006; 25: 7381-7390Crossref PubMed Scopus (150) Google Scholar). Also, it has been shown that gemcitabine induces EGFR internalization and subsequent degradation, which may be a novel mechanism for gemcitabine-mediated cell death (5Feng F.Y. Varambally S. Tomlins S.A. Chun P.Y. Lopez C.A. Li X. Davis M.A. Chinnaiyan A.M. Lawrence T.S. Nyati M.K. Oncogene. 2007; 26: 3431-3439Crossref PubMed Scopus (62) Google Scholar), whereas the activation of p38 MAPK is necessary for gemcitabine-induced cytotoxicity (27Habiro A. Tanno S. Koizumi K. Izawa T. Nakano Y. Osanai M. Mizukami Y. Okumura T. Kohgo Y. Biochem. Biophys. Res. Commun. 2004; 316: 71-77Crossref PubMed Scopus (73) Google Scholar, 28Koizumi K. Tanno S. Nakano Y. Habiro A. Izawa T. Mizukami Y. Okumura T. Kohgo Y. Anticancer Res. 2005; 25: 3347-3353PubMed Google Scholar). Taken together, serine phosphorylation of EGFR via p38 MAPK might be considered a new therapeutic target especially to counter cancer cells of the colon, lung, pancreas, and breast that highly express EGFR.UV radiation from sunlight is sorted by wavelength regions: long wavelength UV-A (320–400 nm), medium wavelength UV-B (280–320 nm), and short wavelength UV-C (200–280 nm). In general, UV-A and UV-B are recognized the major carcinogenic components of sunlight (29Latonen L. Laiho M. Biochim. Biophys. Acta. 2005; 1755: 71-89PubMed Google Scholar). As for UV-C, it is used for studying DNA damage and cellular DNA repair process, although it does not actually exist in earth's surface because they are filtered out by the atmosphere. Although UV-C has been commonly applied for equipment such as water sterilization, recent studies show the possible application of UV-C against human cancer (30Zwang Y. Yarden Y. EMBO J. 2006; 25: 4195-4206Crossref PubMed Scopus (155) Google Scholar). However, its exact mechanism has not been fully clarified.We have recently reported that the blockade of EGF stimulation significantly suppressed SW480 cell growth, suggesting that EGFR pathway plays an important role in proliferation of colon cancer cells (31Nakashima M. Adachi S. Yasuda I. Yamauchi T. Kozawa O. Moriwaki H. Int. J. Oncol. 2010; 36: 585-592PubMed Google Scholar). Therefore, we herein investigated the effect of UV-C on the down-regulation of EGFR in these cells and found that this induces the internalization and degradation of EGFR that indicates the removal of EGFR from cell membrane, and this action of UV-C can protect colon cancer cells from oncogenic stimulation such as EGF, resulting in cell cycle arrest.DISCUSSIONWhereas UV irradiation reportedly has many effects on skin, including inflammation, immunosuppression, and alterations in the extracellular matrix, in addition to accelerated skin aging (51Norval M. J. Photochem. Photobiol. B. 2001; 63: 28-40Crossref PubMed Scopus (68) Google Scholar), in the present study we demonstrated that UV-C has a potent anti-cancer effect by decreasing EGFR protein level in colon cancer cells. First, we showed that UV-C caused anti-survival and proliferative effects on SW480, HT29, and DLD-1 cells (Fig. 1). Because EGFR activation has been shown to be oncogenic (9Rozakis-Adcock M. McGlade J. Mbamalu G. Pelicci G. Daly R. Li W. Batzer A. Thomas S. Brugge J. Pelicci P.G. Schlessinger J. Pawson T. Nature. 1992; 360: 689-692Crossref PubMed Scopus (825) Google Scholar), and we here showed that colon cancer cell proliferation depended on EGFR signaling (Fig. 1, C–E), we examined the effect of UV-C on the EGFR signaling and found that UV-C induced internalization and subsequent down-regulation of the EGFR and also decreased the protein level of cyclin D1 and phospho-Rb (Fig. 2), suggesting that the anti-cancer effect of UV-C could be due to cell cycle arrest in colon cancer cells.In addition, we showed in Fig. 3 that UV-C caused a marked phosphorylation of EGFR at Ser-1046/7; however, UV-C failed to induce phosphorylation at Tyr-1045, the major c-Cbl binding site, as well as Tyr-1068, the Grb2 adaptor protein-binding site (8Lowenstein E.J. Daly R.J. Batzer A.G. Li W. Margolis B. Lammers R. Ullrich A. Skolnik E.Y. Bar-Sagi D. Schlessinger J. Cell. 1992; 70: 431-442Abstract Full Text PDF PubMed Scopus (1331) Google Scholar), in contrast to the prominent phosphorylation induced by EGF as we have previously shown (25Adachi S. Natsume H. Yamauchi J. Matsushima-Nishiwaki R. Joe A.K. Moriwaki H. Kozawa O. Cancer Lett. 2009; 277: 108-113Crossref PubMed Scopus (30) Google Scholar). Moreover, although UV-C caused activation of p44/p42 MAPK, p38 MAPK, or SAPK/JNK (Fig. 4A), we observed that the inhibition of p38 MAPK suppressed EGFR internalization (Fig. 4, B and C). In addition, p38 MAPK was involved in phosphorylation at Ser-1046/7 (Fig. 4, D–F) and subsequent degradation (Fig. 6) of the EGFR induced by UV-C. Moreover, UV-C-induced activation of p38 MAPK was mediated through TAK-1 (Fig. 5). We also examined the effect of UV-C on apoptosis signal-regulating kinase 1, a MAPK kinase kinase, because apoptosis signal-regulating kinase 1 is activated in response to a variety of stress-related stimuli and activates MKK3, which in turn activate p38 MAPK (52Matsuzawa A. Ichijo H. J. Biochem. 2001; 130: 1-8Crossref PubMed Scopus (155) Google Scholar). However, UV-C had no appreciable effect on phosphorylation of apoptosis signal-regulating kinase 1 at Ser-967 and Thr-845 (data not shown). Furthermore, we found in colon cancer cells that pretreatment with UV-C before EGF stimulation significantly suppressed the phosphorylation of EGFR at tyrosine residues and Akt (Fig. 7), indicating that UV-C can evade cells from oncogenic stimulation of EGF. In addition, as shown in Fig. 8, it seems unlikely that DNA damage is involved in UV-C-induced EGFR down-regulation via p38 MAPK. However, our present findings do not evaluate and cannot completely eliminate the possibility that DNA damage plays a role in UV-C-induced cell cycle arrest.Whereas we have recently reported that the blockade of EGF stimulation significantly suppressed cell growth (31Nakashima M. Adachi S. Yasuda I. Yamauchi T. Kozawa O. Moriwaki H. Int. J. Oncol. 2010; 36: 585-592PubMed Google Scholar), we herein demonstrated that proliferation of colon cancer cells depended on the EGFR kinase activity, thus suggesting that the desensitization of EGFR signaling is a promising target against human colon cancer. In addition, an early work showed that exposure to UV light induced clustering and internalization of cell surface EGFR, and inhibition of clustering or receptor down-regulation attenuates UV responses (53Rosette C. Karin M. Science. 1996; 274: 1194-1197Crossref PubMed Scopus (941) Google Scholar), which is consistent with our present findings that internalization and subsequent degradation of EGFR induced by UV-C leads to cell cycle arrest of colon cancer cells by causing its phosphorylation at serine residues via p38 MAPK. Because it is generally understood that tyrosine phosphorylation results in cancer cell proliferation (9Rozakis-Adcock M. McGlade J. Mbamalu G. Pelicci G. Daly R. Li W. Batzer A. Thomas S. Brugge J. Pelicci P.G. Schlessinger J. Pawson T. Nature. 1992; 360: 689-692Crossref PubMed Scopus (825) Google Scholar), these results also suggest the potential availability of UV-C for human colon cancer therapy because UV-C can cause EGFR down-regulation without oncogenic activation (Fig. 3). Moreover, Zwang and Yarden (30Zwang Y. Yarden Y. EMBO J. 2006; 25: 4195-4206Crossref PubMed Scopus (155) Google Scholar) previously reported that abrogating EGFR internalization reduces the efficacy of chemotherapy-induced cell death and EGFR internalization enhances the cytotoxic effect of cisplatin by preventing EGFR-mediated survival signaling, which may underlie interactions between chemotherapy and EGFR-targeting drugs. Therefore, our findings also provide the possibility of a new combination of conventional chemotherapy and UV-C for human colon cancer.Our present study combined with previous findings is summarized in Fig. 9 as follows; when the cells are exposed to EGF stimulation, EGFR undergoes dimerization and tyrosine phosphorylation that directs the cells into cell proliferation (9Rozakis-Adcock M. McGlade J. Mbamalu G. Pelicci G. Daly R. Li W. Batzer A. Thomas S. Brugge J. Pelicci P.G. Schlessinger J. Pawson T. Nature. 1992; 360: 689-692Crossref PubMed Scopus (825) Google Scholar). Subsequently, c-Cbl, a ubiquitin ligase, can bind to EGFR and cause ubiquitination and degradation of the EGFR (10Levkowitz G. Waterman H. Ettenberg S.A. Katz M. Tsygankov A.Y. Alroy I. Lavi S. Iwai K. Reiss Y. Ciechanover A. Lipkowitz S. Yarden Y. Mol. Cell. 1999; 4: 1029-1040Abstract Full Text Full Text PDF PubMed Scopus (829) Google Scholar, 54Levkowitz G. Waterman H. Zamir E. Kam Z. Oved S. Langdon W.Y. Beguinot L. Geiger B. Yarden Y. Genes Dev. 1998; 12: 3663-3674Crossref PubMed Scopus (714) Google Scholar). By contrast, UV-C has little effect on EGFR phosphorylation at tyrosine residues, indicating that adequate dose of UV-C fails to exert the cell growth signals. However, UV-C induced serial phosphorylation of TAK1, MKK3/6, and p38 MAPK and subsequent phosphorylation of EGFR at Ser-1046/7. With time, EGFR molecules are internalized and eventually degraded. Therefore, pretreatment of the cells with UV-C can protect colon cancer cells from oncogenic stimulation such as EGF.We have previously reported that (−)-epigallocatechin gallate, as well as HSP90 inhibitors, causes down-regulation of the EGFR via phosphorylation at Ser-1046/7 through p38 MAPK in human cancer cells (24Adachi S. Shimizu M. Shirakami Y. Yamauchi J. Natsume H. Matsushima-Nishiwaki R. To S. Weinstein I.B. Moriwaki H. Kozawa O. Carcinogenesis. 2009; 30: 1544-1552Crossref PubMed Scopus (85) Google Scholar, 26Adachi S. Yasuda I. Nakashima M. Yamauchi T. Yamauchi J. Natsume H. Moriwaki H. Kozawa O. Oncol. Rep. 2010; 23: 1709-1714Crossref PubMed Scopus (18) Google Scholar). Additionally, accumulating evidence shows that activation of p38 MAPK has an inhibitory effect on tumorigenesis (55Lavoie J.N. L'Allemain G. Brunet A. Müller R. Pouysségur J. J. Biol. Chem. 1996; 271: 20608-20616Abstract Full Text Full Text PDF PubMed Scopus (1075) Google Scholar, 56Kennedy N.J. Cellurale C. Davis R.J. Cancer Cell. 2007; 11: 101-103Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) and that a variety of agents, such as gemcitabine (5Feng F.Y. Varambally S. Tomlins S.A. Chun P.Y. Lopez C.A. Li X. Davis M.A. Chinnaiyan A.M. Lawrence T.S. Nyati M.K. Oncogene. 2007; 26: 3431-3439Crossref PubMed Scopus (62) Google Scholar) and cisplatin (20Winograd-Katz S.E. Levitzki A. Oncogene. 2006; 25: 7381-7390Crossref PubMed Scopus (150) Google Scholar), can also induce activation of p38 MAPK and internalization of EGFR into endosomal vesicles. Moreover, it has previously been reported that the Ser-1046/7 phosphorylation sites act to suppress the oncogenic signal transduction by the wild-type EGFR (21Countaway J.L. McQuilkin P. Gironès N. Davis R.J. J. Biol. Chem. 1990; 265: 3407-3416Abstract Full Text PDF PubMed Google Scholar, 23Theroux S.J. Latour D.A. Stanley K. Raden D.L. Davis R.J. J. Biol. Chem. 1992; 267: 16620-16626Abstract Full Text PDF PubMed Google Scholar). Hence, our present findings might provide a new therapeutic strategy for human colon cancer, although further investigations are necessary to elucidate the mechanism underlying EGFR down-regulation via its phosphorylation at serine residues.Regarding our concern of how these findings would be translated into the clinic, the current limitation of the present study is the lack of practical tools to deliver UV-C irradiation onto the colon cancer tissue in the human body. Our hypothetical approaches would include a combination of extracorporeal generator and fiberscopic transmission of UV-C through colon endoscope or possible expansion of light emitting diode technique to exert shorter wavelength UV-C than with the currently available UV-A light emitting diode. However, substantial technical advances and some related time would be essentially required before practical application of UV-C for colon cancer management. Moreover, further studies are required to determine whether these effects of UV-C safely occur in vivo.In summary, we found that UV-C induces EGFR down-regulation via p38 MAPK-mediated EGFR phosphorylation at Ser-1046/7. Moreover, our results strongly suggest that UV-C irradiation induces the removal of EGFR from cell surfaces that can evade colon cancer cells from oncogenic stimulation of EGF, resulting in cell cycle arrest in colon cancer cells. IntroductionMembers of the EGF receptor (EGFR) 2The abbreviations used are: EGFREGF receptorUV-Cultraviolet-CMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideRbretinoblastoma proteinTAK-1TGF-β-activated kinase-1JJ/m2BrdUbromodeoxyuridineMKKMAPK kinaseSer-1046/7Ser-1046/1047. family, which are frequently overexpressed in several types of human cancers, including cancers of the lung (1Rusch V. Klimstra D. Venkatraman E. Pisters P.W. Langenfeld J. Dmitrovsky E. Clin. Cancer Res. 1997; 3: 515-522PubMed Google Scholar), head and neck (2Masuda M. Suzui M. Weinstein I.B. Clin. Cancer Res. 2001; 7: 4220-4229PubMed Google Scholar), prostate (3Fong C.J. Sherwood E.R. Mendelsohn J. Lee C. Kozlowski J.M. Cancer Res. 1992; 52: 5887-5892PubMed Google Scholar), breast (4Pianetti S. Guo S. Kavanagh K.T. Sonenshein G.E. Cancer Res. 2002; 62: 652-655PubMed Google Scholar), pancreas (5Feng F.Y. Varambally S. Tomlins S.A. Chun P.Y. Lopez C.A. Li X. Davis M.A. Chinnaiyan A.M. Lawrence T.S. Nyati M.K. Oncogene. 2007; 26: 3431-3439Crossref PubMed Scopus (62) Google Scholar), and colon (6Shimizu M. Deguchi A. Lim J.T. Moriwaki H. Kopelovich L. Weinstein I.B. Clin. Cancer Res. 2005; 11: 2735-2746Crossref PubMed Scopus (298) Google Scholar), have been associated with abnormal growth of these tumors. It is well known that exposure of cells to EGF results in rapid autophosphorylation of EGFR molecules at the cell surface (7Meisenhelder J. Suh P.G. Rhee S.G. Hunter T. Cell. 1989; 57: 1109-1122Abstract Full Text PDF PubMed Scopus (601) Google Scholar, 8Lowenstein E.J. Daly R.J. Batzer A.G. Li W. Margolis B. Lammers R. Ullrich A. Skolnik E.Y. Bar-Sagi D. Schlessinger J. Cell. 1992; 70: 431-442Abstract Full Text PDF PubMed Scopus (1331) Google Scholar, 9Rozakis-Adcock M. McGlade J. Mbamalu G. Pelicci G. Daly R. Li W. Batzer A. Thomas S. Brugge J. Pelicci P.G. Schlessinger J. Pawson T. Nature. 1992; 360: 689-692Crossref PubMed Scopus (825) Google Scholar, 10Levkowitz G. Waterman H. Ettenberg S.A. Katz M. Tsygankov A.Y. Alroy I. Lavi S. Iwai K. Reiss Y. Ciechanover A. Lipkowitz S. Yarden Y. Mol. Cell. 1999; 4: 1029-1040Abstract Full Text Full Text PDF PubMed Scopus (829) Google Scholar), which upon activation lead to cell proliferation, motility, and enhanced survival (11Zandi R. Larsen A.B. Andersen P. Stockhausen M.T. Poulsen H.S. Cell Signal. 2007; 19: 2013-2023Crossref PubMed Scopus (229) Google Scholar). There are several mechanisms by which EGFR becomes oncogenic including: 1) increased EGFR expression levels, 2) autocrine and/or paracrine growth factor loops, 3) heterodimerization with other EGFR family members and cross-talk with heterologous receptor systems, 4) defective receptor down-regulation, and 5) activating mutations (12Arteaga C.L. Oncologist. 2002; 7: 31-39Crossref PubMed Scopus (401) Google Scholar). In clinical trials, increasing evidence shows the efficacy of EGFR-targeted agents, including monoclonal antibodies on the one hand and tyrosine kinase inhibitors on the other (13Milano G. Spano J.P. Leyland-Jones B. Br. J. Cancer. 2008; 99: 1-5Crossref PubMed Scopus (41) Google Scholar).Following activation, the ligand-receptor complexes are internalized and then enter endosomes, where the receptors and their ligands are sorted to various intracellular destinations (14Massie C. Mills I.G. Nat. Rev. Cancer. 2006; 6: 403-409Crossref PubMed Scopus (65) Google Scholar). Thus, some receptors can be recycled back to the cell surface via early endosomes, and others are targeted to late endosomes and lysosomes for proteolytic degradation. There is increasing evidence that not only does receptor internalization act to terminate signaling, but that internalized endosome-associated receptors are also able to stimulate specific signal transduction pathways (15Wiley H.S. Exp. Cell Res. 2003; 284: 78-88Crossref PubMed Scopus (299) Google Scholar, 16Di Fiore P.P. De Camilli P. Cell. 2001; 106: 1-4Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, 17McPherson P.S. Kay B.K. Hussain N.K. Traffic. 2001; 2: 375-384Crossref PubMed Scopus (184) Google Scholar). Some agents that induce ligand-independent internalization and degradation of EGFR, such as the 225 mouse antibody (18Jaramillo M.L. Leon Z. Grothe S. Paul-Roc B. Abulrob A. O'Connor McCourt M. Exp. Cell Res. 2006; 312: 2778-2790Crossref PubMed Scopus (75) Google Scholar) and gemcitabine (5Feng F.Y. Varambally S. Tomlins S.A. Chun P.Y. Lopez C.A. Li X. Davis M.A. Chinnaiyan A.M. Lawrence T.S. Nyati M.K. Oncogene. 2007; 26: 3431-3439Crossref PubMed Scopus (62) Google Scholar), could have promising potential for cancer therapies. By contrast, it has previously been reported that the other factors or agents, such as oxidative stress (19Khan E.M. Heidinger J.M. Levy M. Lisanti M.P. Ravid T. Goldkorn T. J. Biol. Chem. 2006; 281: 14486-14493Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) and cisplatin (20Winograd-Katz S.E. Levitzki A. Oncogene. 2006; 25: 7381-7390Crossref PubMed Scopus (150) Google Scholar), can induce internalization of the EGFR but not degradation. They differ in their effects on the fate of the receptors, downstream signaling, and cell proliferation.Receptor down-regulation is the most prominent regulatory system of EGFR signal attenuation and involves the internalization and subsequent degradation of the activated receptor in the lysosomes. With the current knowledge of the mechanism underlying EGFR down-regulation, this molecular event involves several important phosphorylation sites in EGFR. One is the phosphorylation at Tyr-1045, which provides a docking site for the ubiquitin ligase c-Cbl resulting in ubiquitination of the EGFR (10Levkowitz G. Waterman H. Ettenberg S.A. Katz M. Tsygankov A.Y. Alroy I. Lavi S. Iwai K. Reiss Y. Ciechanover A. Lipkowitz S. Yarden Y. Mol. Cell. 1999; 4: 1029-1040Abstract Full Text Full Text PDF PubMed Scopus (829) Google Scholar). The others are the phosphorylation at serine or threonine residues that is thought to represent a mechanism for attenuation of the receptor tyrosine kinase activity (21Countaway J.L. McQuilkin P. Gironès N. Davis R.J. J. Biol. Chem. 1990; 265: 3407-3416Abstract Full Text PDF PubMed Google Scholar, 22Countaway J.L. Nairn A.C. Davis R.J. J. Biol. Chem. 1992; 267: 1129-1140Abstract Full Text PDF PubMed Google Scholar, 23Theroux S.J. Latour D.A. Stanley K. Raden D.L. Davis R.J. J. Biol. Chem. 1992; 267: 16620-16626Abstract Full Text PDF PubMed Google Scholar). Among the major sites of serine and threonine phosphorylation of the EGFR, it has previously been shown that the Ser-1046/7 phosphorylation site is required for EGFR desensitization in EGF-treated cells (23Theroux S.J. Latour D.A. Stanley K. Raden D.L. Davis R.J. J. Biol. Chem. 1992; 267: 16620-16626Abstract Full Text PDF PubMed Google Scholar). Moreover, mutant EGFR at Ser-1046/7 reportedly causes the inhibition of the EGF-induced endocytosis and down-regulation of cell surface receptors (22Countaway J.L. Nairn A.C. Davis R.J. J. Biol. Chem. 1992; 267: 1129-1140Abstract Full Text PDF PubMed Google Scholar).We have recently reported that phosphorylation of EGFR at serine 1046/7 via activation of p38 MAPK plays a pivotal role in down-regulation of EGFR induced by (−)-epigallocatechin gallate (24Adachi S. Shimizu M. Shirakami Y. Yamauchi J. Natsume H. Matsushima-Nishiwaki R. To S. Weinstein I.B. Moriwaki H. Kozawa O. Carcinogenesis. 2009; 30: 1544-1552Crossref PubMed Scopus (85) Google Scholar), anisomycin (25Adachi S. Natsume H. Yamauchi J. Matsushima-Nishiwaki R. Joe A.K. Moriwaki H. Kozawa O. Cancer Lett. 2009; 277: 108-113Crossref PubMed Scopus (30) Google Scholar), and HSP90 inhibitor (26Adachi S. Yasuda I. Nakashima M. Yamauchi T. Yamauchi J. Natsume H. Moriwaki H. Kozawa O. Oncol. Rep. 2010; 23: 1709-1714Crossref PubMed Scopus (18) Google Scholar). Moreover, there is an evidence that cisplatin also induces EGFR internalization, which is mediated by p38 MAPK-dependent phosphorylation of the receptor at threonine 669 (20Winograd-Katz S.E. Levitzki A. Oncogene. 2006; 25: 7381-7390Crossref PubMed Scopus (150) Google Scholar). Also, it has been shown that gemcitabine induces EGFR internalization and subsequent degradation, which may be a novel mechanism for gemcitabine-mediated cell death (5Feng F.Y. Varambally S. Tomlins S.A. Chun P.Y. Lopez C.A. Li X. Davis M.A. Chinnaiyan A.M. Lawrence T.S. Nyati M.K. Oncogene. 2007; 26: 3431-3439Crossref PubMed Scopus (62) Google Scholar), whereas the activation of p38 MAPK is necessary for gemcitabine-induced cytotoxicity (27Habiro A. Tanno S. Koizumi K. Izawa T. Nakano Y. Osanai M. Mizukami Y. Okumura T. Kohgo Y. Biochem. Biophys. Res. Commun. 2004; 316: 71-77Crossref PubMed Scopus (73) Google Scholar, 28Koizumi K. Tanno S. Nakano Y. Habiro A. Izawa T. Mizukami Y. Okumura T. Kohgo Y. Anticancer Res. 2005; 25: 3347-3353PubMed Google Scholar). Taken together, serine phosphorylation of EGFR via p38 MAPK might be considered a new therapeutic target especially to counter cancer cells of the colon, lung, pancreas, and breast that highly express EGFR.UV radiation from sunlight is sorted by wavelength regions: long wavelength UV-A (320–400 nm), medium wavelength UV-B (280–320 nm), and short wavelength UV-C (200–280 nm). In general, UV-A and UV-B are recognized the major carcinogenic components of sunlight (29Latonen L. Laiho M. Biochim. Biophys. Acta. 2005; 1755: 71-89PubMed Google Scholar). As for UV-C, it is used for studying DNA damage and cellular DNA repair process, although it does not actually exist in earth's surface because they are filtered out by the atmosphere. Although UV-C has been commonly applied for equipment such as water sterilization, recent studies show the possible application of UV-C against human cancer (30Zwang Y. Yarden Y. EMBO J. 2006; 25: 4195-4206Crossref PubMed Scopus (155) Google Scholar). However, its exact mechanism has not been fully clarified.We have recently reported that the blockade of EGF stimulation significantly suppressed SW480 cell growth, suggesting that EGFR pathway plays an important role in proliferation of colon cancer cells (31Nakashima M. Adachi S. Yasuda I. Yamauchi T. Kozawa O. Moriwaki H. Int. J. Oncol. 2010; 36: 585-592PubMed Google Scholar). Therefore, we herein investigated the effect of UV-C on the down-regulation of EGFR in these cells and found that this induces the internalization and degradation of EGFR that indicates the removal of EGFR from cell membrane, and this action of UV-C can protect colon cancer cells from oncogenic stimulation such as EGF, resulting in cell cycle arrest.
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