- Email: [email protected]
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Ancona E. Oesophageal resection for high-grade dysplasia in Barrett’s oesophagus. Br J Surg 2000;87:1102–1105. Levine DS, Haggitt RC, Blount PL, Rabinovitch PS, Rusch VW, Reid BJ. An endoscopic biopsy protocol can differentiate highgrade dysplasia from early adenocarcinoma in Barrett’s esophagus. Gastroenterology 1993;105:40 –50. Hameeteman W, Tytgat GNJ, Houthoff HJ, van den Tweel JG. Barrett’s esophagus: development of dysplasia and adenocarcinoma. Gastroenterology 1989;96:1249 –1256. Weston AP, Sharma Prateek S, Topalovski M, Richards R, Cherian R, Dixon A. Long-term follow-up of Barrett’s high-grade dysplasia. Am J Gastroenterol 2000;95:1888 –1893. Reid BJ, Levine DS, Longton G, Blount PL, Rabinovitch PS. Predictors of progression to cancer in Barrett’s esophagus: baseline histology and flow cytometry identify low- and high-risk patient subsets. Am J Gastroenterol 2000;95:1669 –1676. Overholt BF, Panjehpour M, Haydek JM. Photodynamic therapy for Barrett’s esophagus: follow-up in 100 patients. Gastrointest Endosc 1999;49:1–7. Ell C, May A, Gossner L, Pech O, Gunter E, Mayer G, Henrich R, Vieth M, Muller H, Seitz G, Stolte M. Endoscopic mucosal resection of early cancer and high-grade dysplasia in Barrett’s esophagus. Gastroenterology 2000;118:670 – 677. Stein HJ. Esophageal cancer: screening and surveillance. Results of a consensus conference held at the VIth World Congress of the International Society for Diseases of the Esophagus. Dis Esophagus 1996;9(Suppl 1):3–19. Levine DS. Management of dysplasia in the columnar-lined esophagus. Gastroenterol Clin North Am 1997;26:613– 634. Buttar NS, Wang KK, Sebo TJ, Riehle DM, Krishnadath KK, Lutzke LS, Anderson MA, Patterson TM, Burgart LJ. Does the extent of high grade dysplasia in Barrett’s esophagus matter? Gastroenterology 2001:120;1630 –1639. Schnell TG, Sontag SJ, Chejfec G, Aranha G, Metz A, O’Connell S,
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20.
21.
22. 23.
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Sonnenberg A. Long-term non-surgical management of Barrett’s esophagus with high-grade dysplasia. Gastroenterology 2001; 120:1607–1619. Katz D, Rothstein R, Schned A, Dunn J, Seaver K, Antonioli D. The development of dysplasia and adenocarcinoma during endoscopic surveillance of Barrett’s esophagus. Am J Gastroenterol 1998;93:536 –541. O’Connor JB, Falk GW, Richter JE. The incidence of adenocarcinoma and dysplasia in Barrett’s esophagus. Report on the Cleveland Clinic Barrett’s esophagus registry. Am J Gastroenterol 1999;94:2037–2042. Ershler WB. The change in aggressiveness of neoplasms with age. Geriatrics 1987;42:99 –103. Stepp H, Stroka R, Baumgartner R. Fluorescence endoscopy of gastrointestinal diseases: basic principles, techniques, and clinical experience. Endoscopy 1998;30:379 –386. Wallace MB, Perelman LT, Backman V, Crawford JM, Fitzmaurice M, Seiler M, Badizadegan K, Shields SJ, Itzkan I, Dasari RR, Van Dam J, Feld MS. Endoscopic detection of dysplasia in patients with Barrett’s esophagus using light-scattering spectroscopy. Gastroenterology 2000;119:677– 682. Sampliner RE and The Practice Parameters Committee of the American College of Gastroenterology. Practice guidelines on the diagnosis, surveillance, and therapy of Barrett’s esophagus. Am J Gastroenterol 1998;93:1028 –1032.
Address requests for reprints to: Stuart Jon Spechler, M.D., Chief, Division of Gastroenterology (111B1), Dallas VA Medical Center, 4500 South Lancaster Road, Dallas, Texas 75216. e-mail: [email protected]; fax: (214) 857-1571. © 2001 by the American Gastroenterological Association 0016-5085/01/$35.00 doi:10.1053/gast.2001.25291
Protein Kinase C Isozymes in Colon Carcinogenesis: Guilt by Omission See article on page 1700.
he discovery almost 20 years ago that protein kinase C (PKC) is the major cellular receptor for the phorbol ester class of tumor promoters1 provided the first link between PKC signal transduction and carcinogenesis and prompted extensive investigation into the structure, regulation, and function of the enzyme in normal and neoplastic cells. Molecular cloning studies revealed that PKC is in fact a family of at least 10 distinct isozymes (␣, I, II, ␥, ␦, ⑀, , , , and ), which share the same basic structure but differ with respect to activator and cofactor requirements, substrate specificity, tissue expression, and subcellular distribution.2,3 Extensive functional studies have indicated that individual PKC isozymes play specialized roles in cell signaling and control specific cellular responses. The demonstration that members of
T
the PKC family are involved in regulation of 3 key cellular processes that are disrupted in malignant cells, i.e., cell growth/cell cycle progression,4 differentiation,5 and apoptosis,6 together with evidence for changes in the expression and/or activity of PKC isozymes in a variety of malignancies, has provided additional support for the involvement of these molecules in neoplastic transformation. However, despite a large amount of literature on the subject, understanding of the contribution of altered PKC isozyme signaling to the carcinogenic process remains limited. One of the systems in which the expression, activation, and function of individual PKC isozymes has been most extensively studied is the epithelial lining of the intestinal tract.4,7–14 Enterocytes and colonocytes express multiple members of the PKC family, including PKC ␣, I, II, ␦, ⑀, , , , and , suggesting that this enzyme
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system plays an important role in maintenance of normal intestinal homeostasis. Although individual isozymes exhibit distinct patterns of subcellular distribution in intestinal cells,7,12 morphologic and biochemical analysis has shown that all of these molecules are predominantly expressed/activated in cells of the postmitotic compartment.7,9,11,12,14,15 In normal colonic mucosa, for example, PKC isozyme expression follows an increasing gradient from the base of the crypt to the mucosal surface, with PKC isozyme proteins detected mainly in the nonproliferating and terminally differentiated cells.11,12,15 This finding, together with evidence that PKC isozymes undergo marked changes indicative of activation (i.e., increased membrane association/altered solubility properties16) at specific transition points associated with cell growth cessation and differentiation,7,12 suggests that a major function of member(s) of the PKC family is the regulation of postmitotic events during intestinal epithelial self-renewal. Consistent with this notion, recent studies using a nontransformed intestinal crypt cell line showed that PKC signaling can mediate a coordinated program of cell cycle withdrawal in intestinal cells, involving rapid down-regulation of D-type cyclins and increased expression of Cip/Kip cyclin-dependent kinase inhibitors.10 Evidence from studies in other nontransformed systems including keratinocytes,17,18 mammary epithelial cells,19 and esophageal epithelium20 indicates that PKC-dependent pathways may have a widespread function in negative control of cell growth and induction of differentiation. Intestinal neoplasms have also been extensively characterized for changes in PKC activity and PKC isozyme expression. Early studies by Weinstein et al.21 showed that human colonic neoplasms express reduced levels of total PKC activity relative to normal colonic mucosa, a finding that was subsequently confirmed by several other groups.22 Similar findings have been reported in experimental models of colon cancer in rats.23 Decreased levels of PKC activity have also been observed in preneoplastic colonic mucosa24 and in colonic adenomas,8,22,25 indicating that alterations in PKC isozyme regulation occur early in the multistage process of colon carcinogenesis. Although there is some discrepancy among different reports in the literature, a picture is beginning to emerge regarding the specific PKC isozymes that are altered during intestinal neoplastic progression. A number of studies have shown decreased abundance of PKC ␣, I, ␦, ⑀, , and/or in human and rodent colonic tumors,8,12,13,15,26 –29; PKC ␣, I, and seem to be lost early during intestinal carcinogenesis because their expression is markedly reduced in adenomas of the APCmin
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mouse.30 Interestingly, while the majority of PKC family members seem to be down-regulated during intestinal tumor development, elevated levels of PKC II protein have been detected in several experimental models of colon cancer, both in preneoplastic lesions and in colonic tumors.13,28,29 Thus, PKC II and other members of the PKC family may contribute in opposing ways to the development of intestinal neoplasms. Although these findings clearly show that the multistage process of intestinal carcinogenesis is associated with complex alterations in PKC isozyme activity and expression, the contribution of these changes to tumor progression in this tissue remains largely undefined. In this issue of GASTROENTEROLOGY, Cerda et al.31 provide direct evidence for a role of altered PKC ␦ expression in the neoplastic phenotype of colon carcinoma cells. Reduced expression of PKC ␦ has been detected in primary human and rat colonic neoplasms,8,13,32 as well as several colon adenocarcinoma cell lines (e.g., CaCo-2,31 HCT-116, DLD-1, and HCT-8; Bateman and Black, unpublished data, October, 2000). Cerda et al.31 show that increased expression of PKC ␦ in CaCo-2 colon adenocarcinoma cells decreases anchoragedependent and -independent growth, enhances differentiation, and limits the survival of these cells. These responses are consistent with accumulating evidence that PKC ␦ plays a growth-inhibitory,33,34 differentiation-inducing,34 and/or proapoptotic35,36 role in a variety of nonintestinal systems. That PKC ␦ may play a role in these processes in the normal intestine is supported by the demonstration that this isozyme is predominantly expressed/activated in postmitotic colonocytes of the upper crypts and surface mucosa11,12,15 and in functional enterocytes of the villus.7 Thus, PKC ␦ is appropriately positioned to play a role(s) in postmitotic and/or proapoptotic events in the intestinal epithelium in situ. The notion that PKC ␦ may play a tumor-suppressive role in human colon adenocarcinoma cells is supported by findings in other tumor systems. For example, expression of constitutively active PKC ␦ was shown to inhibit colony formation of c-Ha-ras–transformed NIH 3T3 cells,37 inhibition of PKC ␦ enhanced the transformation of c-src overexpressing rat fibroblasts,38 overexpression of PKC ␦ was shown to reverse the malignant phenotype of c-src– transformed rat colonic epithelial cells,39 and targeted overexpression of epitope-tagged PKC ␦ in mouse epidermis dramatically suppressed skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate.40 In skin, it is notable that PKC ␦ levels are significantly reduced in papillomas,40 suggesting that decreased PKC ␦ protein facilitates papilloma formation. Together, these data sug-
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gest that PKC ␦ is a potent suppressor of malignant transformation. Decreased expression of this molecule may, therefore, be permissive for certain critical steps in the multistage process of colon carcinogenesis, such as enhanced proliferation, acquisition of anchorage-independent growth, incomplete differentiation, or reduced apoptosis. The findings presented by Cerda et al.31 add to an increasing body of evidence suggesting that reduced expression of members of the PKC family (including PKC ␣, I, and ) may confer a relative growth advantage to intestinal cells and thus contribute to the process of intestinal carcinogenesis. In this regard, PKC ␣, which has been shown to mediate a program of cell cycle withdrawal in nontransformed intestinal epithelial cells10 and to suppress growth and promote differentiation in a number of nonintestinal systems (for review, see Black4), seems to be lost early during intestinal tumor development. Expression of PKC ␣ protein is reduced in aberrant crypt foci of azoxymethane-treated mice29 and markedly down-regulated in APCmin mouse adenomas30 (Ehsanullah and Black, unpublished results, July, 1999), chemically induced rat and mouse colonic tumors,13,28,29 and sporadic human colon adenocarcinomas.8,12,15 In a recent study that also used the Caco-2 colon adenocarcinoma cell line as a model system,41 Brasitus et al. showed that reduced expression of PKC ␣ results in enhanced cellular proliferation, decreased differentiation, and a more aggressive transformed phenotype. In contrast, increased expression of this isozyme suppressed the transformed phenotype of CaCo-2 cells in vitro and in vivo. Thus, as observed with PKC ␦, diminished activity of PKC ␣– dependent signaling pathways seems to contribute to alterations in growth control/differentiation associated with the development of colonic neoplasia. Loss of PKC I and PKC may also contribute to intestinal carcinogenesis. Although PKC I expression is modestly decreased in aberrant crypt foci,29 it is markedly reduced in APCmin mouse adenomas30 and in azoxymethane-induced colon carcinomas in mice.29 Early studies by Weinstein et al.42 showed that overexpression of PKC I in HT-29 colon carcinoma cells increases cell doubling time, inhibits anchorage-independent cell growth, and markedly reduces the tumorigenicity of these cells in nude mice. These findings led Weinstein et al. to suggest for the first time that, in some tumors, PKC can act as a tumor suppressor gene. Consistent with a role of PKC I in negative regulation of cell proliferation, increased expression of this isozyme was also found to suppress the growth of SW480 colon carcinoma cells.43 Loss of PKC may also play a permissive role in
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intestinal tumor development; PKC is predominantly expressed/activated in postmitotic enterocytes7 and colonocytes,11,12 suggesting a role in regulation of postmitotic events (e.g., cell growth arrest, differentiation) in these tissues. The protein is lost from APCmin mouse adenomas,30 as well as from rat13 and human colon adenocarcinomas.8 Notably, preservation of PKC expression seems to play an important role in the chemopreventive effects of several structurally unrelated agents (i.e., the bile acid ursodeoxycholate,13 the nonsteroidal anti-inflammatory drug piroxicam,44 and a vitamin D3 analogue45) in the azoxymethane-induced model of colon carcinogenesis. Taken together, findings from a number of laboratories suggest that PKC ␣, I, ␦, and play key roles in maintaining the balance between proliferation, differentiation, and apoptosis in the normal intestine. Because these PKC isozymes seem to limit the growth and/or survival of intestinal cells, loss of one or more of these molecules is likely to confer properties to these cells that are permissive for the development of intestinal neoplasms. The available data suggest that reduced expression of PKC ␣, I, and perhaps occurs early during intestinal carcinogenesis, and that PKC ␦ expression is lost at a later stage. Impaired activity of the signaling pathways in which these potential tumor suppressor molecules participate, together with enhanced activity of potential tumor promoters such as PKC II,14,29 are likely to profoundly alter the biology of intestinal cells, contributing to the unregulated growth, incomplete differentiation, and increased survival characteristic of transformed cells. Important challenges for the future include elucidation of the specific signaling cascades/ molecular targets that are controlled by these kinases and determination of the mechanisms underlying the dramatic changes in PKC isozyme expression that occur during intestinal tumor development. Such understanding is likely to point to novel targets for therapeutic intervention in the management of intestinal malignancies. JENNIFER D. BLACK Department of Pharmacology and Therapeutics Roswell Park Cancer Institute Buffalo, New York
References 1. Castagna M, Takai Y, Kaibuchi K, Sano K, Kikkawa U, Nishizuka Y. Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters. J Biol Chem 1982;257:7847–7851. 2. Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature 1984;308:693– 698.
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3. Dekker LV, Parker PJ. Protein kinase C: a question of specificity. Trends Biochem Sci 1994;19:73–77. 4. Black JD. Protein kinase C-mediated regulation of the cell cycle. Front Biosci 2000;5:D406 –D423. 5. Clemens MJ, Trayner I, Menaya J. The role of protein kinase C isoenzymes in the regulation of cell proliferation and differentiation. J Cell Sci 1992;103:881– 887. 6. Musashi M, Ota S, Shiroshita N. The role of protein kinase C isoforms in cell proliferation and apoptosis. Int J Hematol 2000; 72:12–19. 7. Saxon ML, Zhao X, Black JD. Activation of protein kinase C isozymes is associated with post-mitotic events in intestinal epithelial cells in situ. J Cell Biol 1994;126:747–763. 8. Kahl-Rainer P, Karner-Hanusch J, Weiss W, Marian B. Five of six protein kinase C isoenzymes present in normal mucosa show reduced protein levels during tumor development in the human colon. Carcinogenesis 1994;15:779 –782. 9. Frey MR, Saxon ML, Zhao X, Rollins A, Evans SS, Black JD. Protein kinase C isozyme-mediated cell cycle arrest involves induction of p21(waf1/cip1) and p27(kip1) and hypophosphorylation of the retinoblastoma protein in intestinal epithelial cells. J Biol Chem 1997;272:9424 –9435. 10. Frey MR, Clark JA, Leontieva O, Uronis JM, Black AR, Black JD. Protein Kinase C Signaling Mediates a Program of Cell Cycle Withdrawal in the Intestinal Epithelium. J Cell Biol 2000;151: 763–778. 11. Jiang YH, Aukema HM, Davidson LA, Lupton JR, Chapkin RS. Localization of protein kinase C isozymes in rat colon. Cell Growth Differ 1995;6:1381–1386. 12. Verstovsek G, Byrd A, Frey MR, Petrelli NJ, Black JD. Colonocyte differentiation is associated with increased expression and altered distribution of protein kinase C isozymes. Gastroenterology 1998;115:75– 85. 13. Wali RK, Frawley BP Jr, Hartmann S, Roy HK, Khare S, ScaglioneSewell BA, Earnest DL, Sitrin MD, Brasitus TA, Bissonnette M. Mechanism of action of chemoprotective ursodeoxycholate in the azoxymethane model of rat colonic carcinogenesis: potential roles of protein kinase C-alpha, -beta II, and -zeta. Cancer Res 1995;55:5257–5264. 14. Murray NR, Davidson LA, Chapkin RS, Clay Gustafson W, Schattenberg DG, Fields AP. Overexpression of protein kinase C betaII induces colonic hyperproliferation and increased sensitivity to colon carcinogenesis. J Cell Biol 1999;145:699 –711. 15. Kahl-Rainer P, Sedivy R, Marian B. Protein kinase C tissue localization in human colonic tumors suggests a role for adenoma growth control. Gastroenterology 1996;110:1753–1759. 16. Kraft AS, Anderson WB. Phorbol esters increase the amount of Ca2⫹, phospholipid-dependent protein kinase associated with plasma membrane. Nature 1983;301:621– 623. 17. Lee YS, Yuspa SH, Dlugosz AA. Differentiation of cultured human epidermal keratinocytes at high cell densities is mediated by endogenous activation of the protein kinase C signaling pathway. J Invest Dermatol 1998;111:762–766. 18. Ohba M, Ishino K, Kashiwagi M, Kawabe S, Chida K, Huh NH, Kuroki T. Induction of differentiation in normal human keratinocytes by adenovirus-mediated introduction of the eta and delta isoforms of protein kinase C. Mol Cell Biol 1998;18:5199 – 5207. 19. Masso-Welch PA, Verstovsek G, Ip MM. Alterations in the expression and localization of protein kinase C isoforms during mammary gland differentiation. Eur J Cell Biol 1999;78:497–510. 20. Osada S, Hashimoto Y, Nomura S, Kohno Y, Chida K, Tajima O, Kubo K, Akimoto K, Koizumi H, Kitamura Y. Predominant expression of nPKC eta, a Ca(2⫹)-independent isoform of protein kinase C in epithelial tissues, in association with epithelial differentiation. Cell Growth Differ 1993;4:167–175. 21. Guillem JG, O’Brian CA, Fitzer CJ, Forde KA, LoGerfo P, Treat M,
EDITORIALS
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
1871
Weinstein IB. Altered levels of protein kinase C and Ca2⫹-dependent protein kinases in human colon carcinomas. Cancer Res 1987;47:2036 –2039. Kopp R, Noelke B, Sauter G, Schildberg FW, Paumgartner G, Pfeiffer A. Altered protein kinase C activity in biopsies of human colonic adenomas and carcinomas. Cancer Res 1991;51:205– 210. Wali RK, Baum CL, Bolt MJ, Dudeja PK, Sitrin MD, Brasitus TA. Down-regulation of protein kinase C activity in 1,2-dimethylhydrazine- induced rat colonic tumors. Biochim Biophys Acta 1991; 1092:119 –123. Baum CL, Wali RK, Sitrin MD, Bolt MJ, Brasitus TA. 1,2-Dimethylhydrazine–induced alterations in protein kinase C activity in the rat preneoplastic colon. Cancer Res 1990;50:3915–3920. Kusunoki M, Sakanoue Y, Hatada T, Yanagi H, Yamamura T, Utsunomiya J. Protein kinase C activity in human colonic adenoma and colorectal carcinoma. Cancer 1992;69:24 –30. Doi S, Goldstein D, Hug H, Weinstein IB. Expression of multiple isoforms of protein kinase C in normal human colon mucosa and colon tumors and decreased levels of protein kinase C beta and eta mRNAs in the tumors. Mol Carcinog 1994;11:197–203. Kuranami M, Powell CT, Hug H, Zeng Z, Cohen AM, Guillem JG. Differential expression of protein kinase C isoforms in human colorectal cancers. J Surg Res 1995;58:233–239. Craven PA, DeRubertis FR. Alterations in protein kinase C in 1,2-dimethylhydrazine induced colonic carcinogenesis. Cancer Res 1992;52:2216 –2221. Gokmen-Polar Y, Murray NR, Velasco MA, Gatalica Z, Fields AP. Elevated protein kinase C betaII is an early promotive event in colon carcinogenesis. Cancer Res 2001;61:1375–1381. Klein IK, Ritland SR, Burgart LJ, Ziesmer SC, Roche PC, Gendler SJ, et al. Adenoma-specific alterations of protein kinase C isozyme expression in Apc(MIN) mice. Cancer Res 2000;60: 2077–2080. Cerda SR, Bissonnette M, Scaglione-Sewell B, Lyons MR, Khare S, Mustafi R, Brasitus TA. Protein kinase C ␦ inhibits anchoragedependent and -independent growth, enhances differentiation, and increases apoptosis in CaCo-2 cells. Gastroenterology 2001;120:1700 –1712. Craven PA, DeRubertis FR. Loss of protein kinase C delta isozyme immunoreactivity in human adenocarcinomas. Dig Dis Sci 1994; 39:481– 489. Watanabe T, Ono Y, Taniyama Y, Hazama K, Igarashi K, Ogita K, Kikkawa U, Nishizuka Y. Cell division arrest induced by phorbol ester in CHO cells overexpressing protein kinase C delta subspecies. Proc Natl Acad Sci U S A 1992;89:10159 –10163. Mischak H, Pierce JH, Goodnight J, Kazanietz MG, Blumberg PM, Mushinski JF. Phorbol ester–induced myeloid differentiation is mediated by protein kinase C-alpha and -delta and not by protein kinase C-beta II, - epsilon, -zeta, and -eta. J Biol Chem 1993; 268:20110 –20115. Cross T, Griffiths G, Deacon E, Sallis R, Gough M, Watters D, Lord JM. PKC-delta is an apoptotic lamin kinase. Oncogene 2000;19: 2331–2337. Denning MF, Wang Y, Nickoloff BJ, Wrone-Smith T. Protein kinase C delta is activated by caspase-dependent proteolysis during ultraviolet radiation-induced apoptosis of human keratinocytes. J Biol Chem 1998;273:29995–30002. Hirai S, Izumi Y, Higa K, Kaibuchi K, Mizuno K, Osada S, Suzuki K, Ohno S. Ras-dependent signal transduction is indispensable but not sufficient for the activation of AP1/Jun by PKC delta. Embo J 1994;13:2331–2340. Lu Z, Hornia A, Jiang YW, Zang Q, Ohno S, Foster DA: Tumor promotion by depleting cells of protein kinase C delta. Mol Cell Biol 1997;17:3418 –3428. Perletti GP, Marras E, Concari P, Piccinini F, Tashjian AH Jr. PKC
1872
40.
41.
42.
43.
EDITORIALS
delta acts as a growth and tumor suppressor in rat colonic epithelial cells. Oncogene 1999;18:1251–1256. Reddig PJ, Dreckschmidt NE, Ahrens H, Simsiman R, Tseng CP, Zou J, Oberley TD, Verma AK. Transgenic mice overexpressing protein kinase C delta in the epidermis are resistant to skin tumor promotion by 12-O-tetradecanoylphorbol-13- acetate. Cancer Res 1999; 59:5710 –5718. Scaglione-Sewell B, Abraham C, Bissonnette M, Skarosi SF, Hart J, Davidson NO, Wall RK, Davis BH, Sitrin M, Brasitus TA. Decreased PKC-alpha expression increases cellular proliferation, decreases differentiation, and enhances the transformed phenotype of CaCo-2 cells. Cancer Res 1998;58:1074 –1081. Choi PM, Tchou-Wong KM, Weinstein IB. Overexpression of protein kinase C in HT29 colon cancer cells causes growth inhibition and tumor suppression. Mol Cell Biol 1990;10:4650 – 4657. Goldstein DR, Cacace AM, Weinstein IB. Overexpression of protein kinase C beta 1 in the SW480 colon cancer cell line causes growth suppression. Carcinogenesis 1995;16:1121–1126.
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44. Roy HK, Bissonnette M, Frawley BP Jr, Wali RK, Niedziela SM, Earnest D, Brasitus TA. Selective preservation of protein kinase C-zeta in the chemoprevention of azoxymethane-induced colonic tumors by piroxicam. FEBS Lett 1995;366:143–145. 45. Wali RK, Bissonnette M, Khare S, Aquino B, Niedziela S, Sitrin M, Brasitus TA. Protein kinase C isoforms in the chemopreventive effects of a novel vitamin D3 analogue in rat colonic tumorigenesis. Gastroenterology 1996;111:118 –126.
Address requests for reprints to: Jennifer D. Black, Ph.D., Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, New York 14263. e-mail: [email protected]; fax: (716) 845-8857. © 2001 by the American Gastroenterological Association 0016-5085/01/$35.00 doi:10.1053/gast.2001.25287
Epidermal Growth Factor Receptor Blockade: An Emerging Therapeutic Modality in Gastroenterology See article on page 1713.
t has now been 30 years since the discovery of epidermal growth factor (EGF). The biology of this peptide and its receptor, the epidermal growth factor receptor (EGFR), has long been of special interest to gastroenterologists, perhaps because early studies reported physiologically relevant concentrations of the peptide in human milk and gastrointestinal secretions. An enormous volume of basic scientific literature has been amassed and much of this information has been integrated into a clearer understanding of this ligand/receptor system in normal and diseased gastrointestinal tissues. The rate of discovery proceeds apace. Exciting recent reports in animal models and in human diseases illustrate how aberrant EGF ligand receptor signaling can be modified to therapeutic advantage. It is clear that the potential for further therapeutic strategies is substantial. This commentary provides an overview of the EGF/EGFR axis and reviews selected recent studies, including the report by Mann et al.1 in this issue of GASTROENTEROLOGY, emphasizing the potential utility of inhibitors of this pathway in clinical gastroenterology and gastrointestinal oncology.
I
The EGF Family of Ligands and Receptors The EGF-related peptide family is comprised of polypeptides sharing sequence homology, affinity for the
same receptor, and a similar spectrum of biological activity.2 The mammalian family includes EGF, transforming growth factor ␣ (TGF-␣), amphiregulin (AR), heparin binding epidermal growth factor–like growth factor (HB-EGF), betacellulin (BTC), and epiregulin. Each is synthesized as a propeptide containing a cytoplasmic domain, a transmembrane sequence, and an extracellular domain. ADAM metalloproteases proteolytically release the mature peptide sequence from the extracellular domain. More distantly related EGF-like molecules include glial growth factor, acetylcholine receptor inducing activity, heregulin, and neu differentiation factor (collectively now termed the neuregulins) that also bind to selected EGFR-like molecules. The are 4 members of the mammalian EGFR family. These proteins are membrane-associated receptor tyrosine kinases with a single extracellular binding domain, a transmembrane domain and an intrinsic tyrosine kinase domain in the cytoplasmic tail. The classical EGF receptor is also known as HER1 (human epidermal growth factor receptor 1) or ErbB1, because of homology with the v-erbB oncogene. All closely related EGF peptides bind ErbB1. The remaining 3 receptors are designated ErbB2 (also referred to as Her2 or the neu protooncogene), ErbB3, and ErbB4. c-ErbB2 is unique in that it does not have an identified ligand. Ligand-induced receptor homo- and heterodimerization is a central feature of signaling by the EGFR. The most plausible model for receptor dimerization proposes 2 receptor-binding sites on each monomeric EGF-related
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11.
12.
13.
14.
15.
16.
17. 18.
19.
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Ancona E. Oesophageal resection for high-grade dysplasia in Barrett’s oesophagus. Br J Surg 2000;87:1102–1105. Levine DS, Haggitt RC, Blount PL, Rabinovitch PS, Rusch VW, Reid BJ. An endoscopic biopsy protocol can differentiate highgrade dysplasia from early adenocarcinoma in Barrett’s esophagus. Gastroenterology 1993;105:40 –50. Hameeteman W, Tytgat GNJ, Houthoff HJ, van den Tweel JG. Barrett’s esophagus: development of dysplasia and adenocarcinoma. Gastroenterology 1989;96:1249 –1256. Weston AP, Sharma Prateek S, Topalovski M, Richards R, Cherian R, Dixon A. Long-term follow-up of Barrett’s high-grade dysplasia. Am J Gastroenterol 2000;95:1888 –1893. Reid BJ, Levine DS, Longton G, Blount PL, Rabinovitch PS. Predictors of progression to cancer in Barrett’s esophagus: baseline histology and flow cytometry identify low- and high-risk patient subsets. Am J Gastroenterol 2000;95:1669 –1676. Overholt BF, Panjehpour M, Haydek JM. Photodynamic therapy for Barrett’s esophagus: follow-up in 100 patients. Gastrointest Endosc 1999;49:1–7. Ell C, May A, Gossner L, Pech O, Gunter E, Mayer G, Henrich R, Vieth M, Muller H, Seitz G, Stolte M. Endoscopic mucosal resection of early cancer and high-grade dysplasia in Barrett’s esophagus. Gastroenterology 2000;118:670 – 677. Stein HJ. Esophageal cancer: screening and surveillance. Results of a consensus conference held at the VIth World Congress of the International Society for Diseases of the Esophagus. Dis Esophagus 1996;9(Suppl 1):3–19. Levine DS. Management of dysplasia in the columnar-lined esophagus. Gastroenterol Clin North Am 1997;26:613– 634. Buttar NS, Wang KK, Sebo TJ, Riehle DM, Krishnadath KK, Lutzke LS, Anderson MA, Patterson TM, Burgart LJ. Does the extent of high grade dysplasia in Barrett’s esophagus matter? Gastroenterology 2001:120;1630 –1639. Schnell TG, Sontag SJ, Chejfec G, Aranha G, Metz A, O’Connell S,
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22. 23.
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Sonnenberg A. Long-term non-surgical management of Barrett’s esophagus with high-grade dysplasia. Gastroenterology 2001; 120:1607–1619. Katz D, Rothstein R, Schned A, Dunn J, Seaver K, Antonioli D. The development of dysplasia and adenocarcinoma during endoscopic surveillance of Barrett’s esophagus. Am J Gastroenterol 1998;93:536 –541. O’Connor JB, Falk GW, Richter JE. The incidence of adenocarcinoma and dysplasia in Barrett’s esophagus. Report on the Cleveland Clinic Barrett’s esophagus registry. Am J Gastroenterol 1999;94:2037–2042. Ershler WB. The change in aggressiveness of neoplasms with age. Geriatrics 1987;42:99 –103. Stepp H, Stroka R, Baumgartner R. Fluorescence endoscopy of gastrointestinal diseases: basic principles, techniques, and clinical experience. Endoscopy 1998;30:379 –386. Wallace MB, Perelman LT, Backman V, Crawford JM, Fitzmaurice M, Seiler M, Badizadegan K, Shields SJ, Itzkan I, Dasari RR, Van Dam J, Feld MS. Endoscopic detection of dysplasia in patients with Barrett’s esophagus using light-scattering spectroscopy. Gastroenterology 2000;119:677– 682. Sampliner RE and The Practice Parameters Committee of the American College of Gastroenterology. Practice guidelines on the diagnosis, surveillance, and therapy of Barrett’s esophagus. Am J Gastroenterol 1998;93:1028 –1032.
Address requests for reprints to: Stuart Jon Spechler, M.D., Chief, Division of Gastroenterology (111B1), Dallas VA Medical Center, 4500 South Lancaster Road, Dallas, Texas 75216. e-mail: [email protected]; fax: (214) 857-1571. © 2001 by the American Gastroenterological Association 0016-5085/01/$35.00 doi:10.1053/gast.2001.25291
Protein Kinase C Isozymes in Colon Carcinogenesis: Guilt by Omission See article on page 1700.
he discovery almost 20 years ago that protein kinase C (PKC) is the major cellular receptor for the phorbol ester class of tumor promoters1 provided the first link between PKC signal transduction and carcinogenesis and prompted extensive investigation into the structure, regulation, and function of the enzyme in normal and neoplastic cells. Molecular cloning studies revealed that PKC is in fact a family of at least 10 distinct isozymes (␣, I, II, ␥, ␦, ⑀, , , , and ), which share the same basic structure but differ with respect to activator and cofactor requirements, substrate specificity, tissue expression, and subcellular distribution.2,3 Extensive functional studies have indicated that individual PKC isozymes play specialized roles in cell signaling and control specific cellular responses. The demonstration that members of
T
the PKC family are involved in regulation of 3 key cellular processes that are disrupted in malignant cells, i.e., cell growth/cell cycle progression,4 differentiation,5 and apoptosis,6 together with evidence for changes in the expression and/or activity of PKC isozymes in a variety of malignancies, has provided additional support for the involvement of these molecules in neoplastic transformation. However, despite a large amount of literature on the subject, understanding of the contribution of altered PKC isozyme signaling to the carcinogenic process remains limited. One of the systems in which the expression, activation, and function of individual PKC isozymes has been most extensively studied is the epithelial lining of the intestinal tract.4,7–14 Enterocytes and colonocytes express multiple members of the PKC family, including PKC ␣, I, II, ␦, ⑀, , , , and , suggesting that this enzyme
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system plays an important role in maintenance of normal intestinal homeostasis. Although individual isozymes exhibit distinct patterns of subcellular distribution in intestinal cells,7,12 morphologic and biochemical analysis has shown that all of these molecules are predominantly expressed/activated in cells of the postmitotic compartment.7,9,11,12,14,15 In normal colonic mucosa, for example, PKC isozyme expression follows an increasing gradient from the base of the crypt to the mucosal surface, with PKC isozyme proteins detected mainly in the nonproliferating and terminally differentiated cells.11,12,15 This finding, together with evidence that PKC isozymes undergo marked changes indicative of activation (i.e., increased membrane association/altered solubility properties16) at specific transition points associated with cell growth cessation and differentiation,7,12 suggests that a major function of member(s) of the PKC family is the regulation of postmitotic events during intestinal epithelial self-renewal. Consistent with this notion, recent studies using a nontransformed intestinal crypt cell line showed that PKC signaling can mediate a coordinated program of cell cycle withdrawal in intestinal cells, involving rapid down-regulation of D-type cyclins and increased expression of Cip/Kip cyclin-dependent kinase inhibitors.10 Evidence from studies in other nontransformed systems including keratinocytes,17,18 mammary epithelial cells,19 and esophageal epithelium20 indicates that PKC-dependent pathways may have a widespread function in negative control of cell growth and induction of differentiation. Intestinal neoplasms have also been extensively characterized for changes in PKC activity and PKC isozyme expression. Early studies by Weinstein et al.21 showed that human colonic neoplasms express reduced levels of total PKC activity relative to normal colonic mucosa, a finding that was subsequently confirmed by several other groups.22 Similar findings have been reported in experimental models of colon cancer in rats.23 Decreased levels of PKC activity have also been observed in preneoplastic colonic mucosa24 and in colonic adenomas,8,22,25 indicating that alterations in PKC isozyme regulation occur early in the multistage process of colon carcinogenesis. Although there is some discrepancy among different reports in the literature, a picture is beginning to emerge regarding the specific PKC isozymes that are altered during intestinal neoplastic progression. A number of studies have shown decreased abundance of PKC ␣, I, ␦, ⑀, , and/or in human and rodent colonic tumors,8,12,13,15,26 –29; PKC ␣, I, and seem to be lost early during intestinal carcinogenesis because their expression is markedly reduced in adenomas of the APCmin
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mouse.30 Interestingly, while the majority of PKC family members seem to be down-regulated during intestinal tumor development, elevated levels of PKC II protein have been detected in several experimental models of colon cancer, both in preneoplastic lesions and in colonic tumors.13,28,29 Thus, PKC II and other members of the PKC family may contribute in opposing ways to the development of intestinal neoplasms. Although these findings clearly show that the multistage process of intestinal carcinogenesis is associated with complex alterations in PKC isozyme activity and expression, the contribution of these changes to tumor progression in this tissue remains largely undefined. In this issue of GASTROENTEROLOGY, Cerda et al.31 provide direct evidence for a role of altered PKC ␦ expression in the neoplastic phenotype of colon carcinoma cells. Reduced expression of PKC ␦ has been detected in primary human and rat colonic neoplasms,8,13,32 as well as several colon adenocarcinoma cell lines (e.g., CaCo-2,31 HCT-116, DLD-1, and HCT-8; Bateman and Black, unpublished data, October, 2000). Cerda et al.31 show that increased expression of PKC ␦ in CaCo-2 colon adenocarcinoma cells decreases anchoragedependent and -independent growth, enhances differentiation, and limits the survival of these cells. These responses are consistent with accumulating evidence that PKC ␦ plays a growth-inhibitory,33,34 differentiation-inducing,34 and/or proapoptotic35,36 role in a variety of nonintestinal systems. That PKC ␦ may play a role in these processes in the normal intestine is supported by the demonstration that this isozyme is predominantly expressed/activated in postmitotic colonocytes of the upper crypts and surface mucosa11,12,15 and in functional enterocytes of the villus.7 Thus, PKC ␦ is appropriately positioned to play a role(s) in postmitotic and/or proapoptotic events in the intestinal epithelium in situ. The notion that PKC ␦ may play a tumor-suppressive role in human colon adenocarcinoma cells is supported by findings in other tumor systems. For example, expression of constitutively active PKC ␦ was shown to inhibit colony formation of c-Ha-ras–transformed NIH 3T3 cells,37 inhibition of PKC ␦ enhanced the transformation of c-src overexpressing rat fibroblasts,38 overexpression of PKC ␦ was shown to reverse the malignant phenotype of c-src– transformed rat colonic epithelial cells,39 and targeted overexpression of epitope-tagged PKC ␦ in mouse epidermis dramatically suppressed skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate.40 In skin, it is notable that PKC ␦ levels are significantly reduced in papillomas,40 suggesting that decreased PKC ␦ protein facilitates papilloma formation. Together, these data sug-
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gest that PKC ␦ is a potent suppressor of malignant transformation. Decreased expression of this molecule may, therefore, be permissive for certain critical steps in the multistage process of colon carcinogenesis, such as enhanced proliferation, acquisition of anchorage-independent growth, incomplete differentiation, or reduced apoptosis. The findings presented by Cerda et al.31 add to an increasing body of evidence suggesting that reduced expression of members of the PKC family (including PKC ␣, I, and ) may confer a relative growth advantage to intestinal cells and thus contribute to the process of intestinal carcinogenesis. In this regard, PKC ␣, which has been shown to mediate a program of cell cycle withdrawal in nontransformed intestinal epithelial cells10 and to suppress growth and promote differentiation in a number of nonintestinal systems (for review, see Black4), seems to be lost early during intestinal tumor development. Expression of PKC ␣ protein is reduced in aberrant crypt foci of azoxymethane-treated mice29 and markedly down-regulated in APCmin mouse adenomas30 (Ehsanullah and Black, unpublished results, July, 1999), chemically induced rat and mouse colonic tumors,13,28,29 and sporadic human colon adenocarcinomas.8,12,15 In a recent study that also used the Caco-2 colon adenocarcinoma cell line as a model system,41 Brasitus et al. showed that reduced expression of PKC ␣ results in enhanced cellular proliferation, decreased differentiation, and a more aggressive transformed phenotype. In contrast, increased expression of this isozyme suppressed the transformed phenotype of CaCo-2 cells in vitro and in vivo. Thus, as observed with PKC ␦, diminished activity of PKC ␣– dependent signaling pathways seems to contribute to alterations in growth control/differentiation associated with the development of colonic neoplasia. Loss of PKC I and PKC may also contribute to intestinal carcinogenesis. Although PKC I expression is modestly decreased in aberrant crypt foci,29 it is markedly reduced in APCmin mouse adenomas30 and in azoxymethane-induced colon carcinomas in mice.29 Early studies by Weinstein et al.42 showed that overexpression of PKC I in HT-29 colon carcinoma cells increases cell doubling time, inhibits anchorage-independent cell growth, and markedly reduces the tumorigenicity of these cells in nude mice. These findings led Weinstein et al. to suggest for the first time that, in some tumors, PKC can act as a tumor suppressor gene. Consistent with a role of PKC I in negative regulation of cell proliferation, increased expression of this isozyme was also found to suppress the growth of SW480 colon carcinoma cells.43 Loss of PKC may also play a permissive role in
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intestinal tumor development; PKC is predominantly expressed/activated in postmitotic enterocytes7 and colonocytes,11,12 suggesting a role in regulation of postmitotic events (e.g., cell growth arrest, differentiation) in these tissues. The protein is lost from APCmin mouse adenomas,30 as well as from rat13 and human colon adenocarcinomas.8 Notably, preservation of PKC expression seems to play an important role in the chemopreventive effects of several structurally unrelated agents (i.e., the bile acid ursodeoxycholate,13 the nonsteroidal anti-inflammatory drug piroxicam,44 and a vitamin D3 analogue45) in the azoxymethane-induced model of colon carcinogenesis. Taken together, findings from a number of laboratories suggest that PKC ␣, I, ␦, and play key roles in maintaining the balance between proliferation, differentiation, and apoptosis in the normal intestine. Because these PKC isozymes seem to limit the growth and/or survival of intestinal cells, loss of one or more of these molecules is likely to confer properties to these cells that are permissive for the development of intestinal neoplasms. The available data suggest that reduced expression of PKC ␣, I, and perhaps occurs early during intestinal carcinogenesis, and that PKC ␦ expression is lost at a later stage. Impaired activity of the signaling pathways in which these potential tumor suppressor molecules participate, together with enhanced activity of potential tumor promoters such as PKC II,14,29 are likely to profoundly alter the biology of intestinal cells, contributing to the unregulated growth, incomplete differentiation, and increased survival characteristic of transformed cells. Important challenges for the future include elucidation of the specific signaling cascades/ molecular targets that are controlled by these kinases and determination of the mechanisms underlying the dramatic changes in PKC isozyme expression that occur during intestinal tumor development. Such understanding is likely to point to novel targets for therapeutic intervention in the management of intestinal malignancies. JENNIFER D. BLACK Department of Pharmacology and Therapeutics Roswell Park Cancer Institute Buffalo, New York
References 1. Castagna M, Takai Y, Kaibuchi K, Sano K, Kikkawa U, Nishizuka Y. Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters. J Biol Chem 1982;257:7847–7851. 2. Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature 1984;308:693– 698.
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3. Dekker LV, Parker PJ. Protein kinase C: a question of specificity. Trends Biochem Sci 1994;19:73–77. 4. Black JD. Protein kinase C-mediated regulation of the cell cycle. Front Biosci 2000;5:D406 –D423. 5. Clemens MJ, Trayner I, Menaya J. The role of protein kinase C isoenzymes in the regulation of cell proliferation and differentiation. J Cell Sci 1992;103:881– 887. 6. Musashi M, Ota S, Shiroshita N. The role of protein kinase C isoforms in cell proliferation and apoptosis. Int J Hematol 2000; 72:12–19. 7. Saxon ML, Zhao X, Black JD. Activation of protein kinase C isozymes is associated with post-mitotic events in intestinal epithelial cells in situ. J Cell Biol 1994;126:747–763. 8. Kahl-Rainer P, Karner-Hanusch J, Weiss W, Marian B. Five of six protein kinase C isoenzymes present in normal mucosa show reduced protein levels during tumor development in the human colon. Carcinogenesis 1994;15:779 –782. 9. Frey MR, Saxon ML, Zhao X, Rollins A, Evans SS, Black JD. Protein kinase C isozyme-mediated cell cycle arrest involves induction of p21(waf1/cip1) and p27(kip1) and hypophosphorylation of the retinoblastoma protein in intestinal epithelial cells. J Biol Chem 1997;272:9424 –9435. 10. Frey MR, Clark JA, Leontieva O, Uronis JM, Black AR, Black JD. Protein Kinase C Signaling Mediates a Program of Cell Cycle Withdrawal in the Intestinal Epithelium. J Cell Biol 2000;151: 763–778. 11. Jiang YH, Aukema HM, Davidson LA, Lupton JR, Chapkin RS. Localization of protein kinase C isozymes in rat colon. Cell Growth Differ 1995;6:1381–1386. 12. Verstovsek G, Byrd A, Frey MR, Petrelli NJ, Black JD. Colonocyte differentiation is associated with increased expression and altered distribution of protein kinase C isozymes. Gastroenterology 1998;115:75– 85. 13. Wali RK, Frawley BP Jr, Hartmann S, Roy HK, Khare S, ScaglioneSewell BA, Earnest DL, Sitrin MD, Brasitus TA, Bissonnette M. Mechanism of action of chemoprotective ursodeoxycholate in the azoxymethane model of rat colonic carcinogenesis: potential roles of protein kinase C-alpha, -beta II, and -zeta. Cancer Res 1995;55:5257–5264. 14. Murray NR, Davidson LA, Chapkin RS, Clay Gustafson W, Schattenberg DG, Fields AP. Overexpression of protein kinase C betaII induces colonic hyperproliferation and increased sensitivity to colon carcinogenesis. J Cell Biol 1999;145:699 –711. 15. Kahl-Rainer P, Sedivy R, Marian B. Protein kinase C tissue localization in human colonic tumors suggests a role for adenoma growth control. Gastroenterology 1996;110:1753–1759. 16. Kraft AS, Anderson WB. Phorbol esters increase the amount of Ca2⫹, phospholipid-dependent protein kinase associated with plasma membrane. Nature 1983;301:621– 623. 17. Lee YS, Yuspa SH, Dlugosz AA. Differentiation of cultured human epidermal keratinocytes at high cell densities is mediated by endogenous activation of the protein kinase C signaling pathway. J Invest Dermatol 1998;111:762–766. 18. Ohba M, Ishino K, Kashiwagi M, Kawabe S, Chida K, Huh NH, Kuroki T. Induction of differentiation in normal human keratinocytes by adenovirus-mediated introduction of the eta and delta isoforms of protein kinase C. Mol Cell Biol 1998;18:5199 – 5207. 19. Masso-Welch PA, Verstovsek G, Ip MM. Alterations in the expression and localization of protein kinase C isoforms during mammary gland differentiation. Eur J Cell Biol 1999;78:497–510. 20. Osada S, Hashimoto Y, Nomura S, Kohno Y, Chida K, Tajima O, Kubo K, Akimoto K, Koizumi H, Kitamura Y. Predominant expression of nPKC eta, a Ca(2⫹)-independent isoform of protein kinase C in epithelial tissues, in association with epithelial differentiation. Cell Growth Differ 1993;4:167–175. 21. Guillem JG, O’Brian CA, Fitzer CJ, Forde KA, LoGerfo P, Treat M,
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22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
1871
Weinstein IB. Altered levels of protein kinase C and Ca2⫹-dependent protein kinases in human colon carcinomas. Cancer Res 1987;47:2036 –2039. Kopp R, Noelke B, Sauter G, Schildberg FW, Paumgartner G, Pfeiffer A. Altered protein kinase C activity in biopsies of human colonic adenomas and carcinomas. Cancer Res 1991;51:205– 210. Wali RK, Baum CL, Bolt MJ, Dudeja PK, Sitrin MD, Brasitus TA. Down-regulation of protein kinase C activity in 1,2-dimethylhydrazine- induced rat colonic tumors. Biochim Biophys Acta 1991; 1092:119 –123. Baum CL, Wali RK, Sitrin MD, Bolt MJ, Brasitus TA. 1,2-Dimethylhydrazine–induced alterations in protein kinase C activity in the rat preneoplastic colon. Cancer Res 1990;50:3915–3920. Kusunoki M, Sakanoue Y, Hatada T, Yanagi H, Yamamura T, Utsunomiya J. Protein kinase C activity in human colonic adenoma and colorectal carcinoma. Cancer 1992;69:24 –30. Doi S, Goldstein D, Hug H, Weinstein IB. Expression of multiple isoforms of protein kinase C in normal human colon mucosa and colon tumors and decreased levels of protein kinase C beta and eta mRNAs in the tumors. Mol Carcinog 1994;11:197–203. Kuranami M, Powell CT, Hug H, Zeng Z, Cohen AM, Guillem JG. Differential expression of protein kinase C isoforms in human colorectal cancers. J Surg Res 1995;58:233–239. Craven PA, DeRubertis FR. Alterations in protein kinase C in 1,2-dimethylhydrazine induced colonic carcinogenesis. Cancer Res 1992;52:2216 –2221. Gokmen-Polar Y, Murray NR, Velasco MA, Gatalica Z, Fields AP. Elevated protein kinase C betaII is an early promotive event in colon carcinogenesis. Cancer Res 2001;61:1375–1381. Klein IK, Ritland SR, Burgart LJ, Ziesmer SC, Roche PC, Gendler SJ, et al. Adenoma-specific alterations of protein kinase C isozyme expression in Apc(MIN) mice. Cancer Res 2000;60: 2077–2080. Cerda SR, Bissonnette M, Scaglione-Sewell B, Lyons MR, Khare S, Mustafi R, Brasitus TA. Protein kinase C ␦ inhibits anchoragedependent and -independent growth, enhances differentiation, and increases apoptosis in CaCo-2 cells. Gastroenterology 2001;120:1700 –1712. Craven PA, DeRubertis FR. Loss of protein kinase C delta isozyme immunoreactivity in human adenocarcinomas. Dig Dis Sci 1994; 39:481– 489. Watanabe T, Ono Y, Taniyama Y, Hazama K, Igarashi K, Ogita K, Kikkawa U, Nishizuka Y. Cell division arrest induced by phorbol ester in CHO cells overexpressing protein kinase C delta subspecies. Proc Natl Acad Sci U S A 1992;89:10159 –10163. Mischak H, Pierce JH, Goodnight J, Kazanietz MG, Blumberg PM, Mushinski JF. Phorbol ester–induced myeloid differentiation is mediated by protein kinase C-alpha and -delta and not by protein kinase C-beta II, - epsilon, -zeta, and -eta. J Biol Chem 1993; 268:20110 –20115. Cross T, Griffiths G, Deacon E, Sallis R, Gough M, Watters D, Lord JM. PKC-delta is an apoptotic lamin kinase. Oncogene 2000;19: 2331–2337. Denning MF, Wang Y, Nickoloff BJ, Wrone-Smith T. Protein kinase C delta is activated by caspase-dependent proteolysis during ultraviolet radiation-induced apoptosis of human keratinocytes. J Biol Chem 1998;273:29995–30002. Hirai S, Izumi Y, Higa K, Kaibuchi K, Mizuno K, Osada S, Suzuki K, Ohno S. Ras-dependent signal transduction is indispensable but not sufficient for the activation of AP1/Jun by PKC delta. Embo J 1994;13:2331–2340. Lu Z, Hornia A, Jiang YW, Zang Q, Ohno S, Foster DA: Tumor promotion by depleting cells of protein kinase C delta. Mol Cell Biol 1997;17:3418 –3428. Perletti GP, Marras E, Concari P, Piccinini F, Tashjian AH Jr. PKC
1872
40.
41.
42.
43.
EDITORIALS
delta acts as a growth and tumor suppressor in rat colonic epithelial cells. Oncogene 1999;18:1251–1256. Reddig PJ, Dreckschmidt NE, Ahrens H, Simsiman R, Tseng CP, Zou J, Oberley TD, Verma AK. Transgenic mice overexpressing protein kinase C delta in the epidermis are resistant to skin tumor promotion by 12-O-tetradecanoylphorbol-13- acetate. Cancer Res 1999; 59:5710 –5718. Scaglione-Sewell B, Abraham C, Bissonnette M, Skarosi SF, Hart J, Davidson NO, Wall RK, Davis BH, Sitrin M, Brasitus TA. Decreased PKC-alpha expression increases cellular proliferation, decreases differentiation, and enhances the transformed phenotype of CaCo-2 cells. Cancer Res 1998;58:1074 –1081. Choi PM, Tchou-Wong KM, Weinstein IB. Overexpression of protein kinase C in HT29 colon cancer cells causes growth inhibition and tumor suppression. Mol Cell Biol 1990;10:4650 – 4657. Goldstein DR, Cacace AM, Weinstein IB. Overexpression of protein kinase C beta 1 in the SW480 colon cancer cell line causes growth suppression. Carcinogenesis 1995;16:1121–1126.
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44. Roy HK, Bissonnette M, Frawley BP Jr, Wali RK, Niedziela SM, Earnest D, Brasitus TA. Selective preservation of protein kinase C-zeta in the chemoprevention of azoxymethane-induced colonic tumors by piroxicam. FEBS Lett 1995;366:143–145. 45. Wali RK, Bissonnette M, Khare S, Aquino B, Niedziela S, Sitrin M, Brasitus TA. Protein kinase C isoforms in the chemopreventive effects of a novel vitamin D3 analogue in rat colonic tumorigenesis. Gastroenterology 1996;111:118 –126.
Address requests for reprints to: Jennifer D. Black, Ph.D., Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, New York 14263. e-mail: [email protected]; fax: (716) 845-8857. © 2001 by the American Gastroenterological Association 0016-5085/01/$35.00 doi:10.1053/gast.2001.25287
Epidermal Growth Factor Receptor Blockade: An Emerging Therapeutic Modality in Gastroenterology See article on page 1713.
t has now been 30 years since the discovery of epidermal growth factor (EGF). The biology of this peptide and its receptor, the epidermal growth factor receptor (EGFR), has long been of special interest to gastroenterologists, perhaps because early studies reported physiologically relevant concentrations of the peptide in human milk and gastrointestinal secretions. An enormous volume of basic scientific literature has been amassed and much of this information has been integrated into a clearer understanding of this ligand/receptor system in normal and diseased gastrointestinal tissues. The rate of discovery proceeds apace. Exciting recent reports in animal models and in human diseases illustrate how aberrant EGF ligand receptor signaling can be modified to therapeutic advantage. It is clear that the potential for further therapeutic strategies is substantial. This commentary provides an overview of the EGF/EGFR axis and reviews selected recent studies, including the report by Mann et al.1 in this issue of GASTROENTEROLOGY, emphasizing the potential utility of inhibitors of this pathway in clinical gastroenterology and gastrointestinal oncology.
I
The EGF Family of Ligands and Receptors The EGF-related peptide family is comprised of polypeptides sharing sequence homology, affinity for the
same receptor, and a similar spectrum of biological activity.2 The mammalian family includes EGF, transforming growth factor ␣ (TGF-␣), amphiregulin (AR), heparin binding epidermal growth factor–like growth factor (HB-EGF), betacellulin (BTC), and epiregulin. Each is synthesized as a propeptide containing a cytoplasmic domain, a transmembrane sequence, and an extracellular domain. ADAM metalloproteases proteolytically release the mature peptide sequence from the extracellular domain. More distantly related EGF-like molecules include glial growth factor, acetylcholine receptor inducing activity, heregulin, and neu differentiation factor (collectively now termed the neuregulins) that also bind to selected EGFR-like molecules. The are 4 members of the mammalian EGFR family. These proteins are membrane-associated receptor tyrosine kinases with a single extracellular binding domain, a transmembrane domain and an intrinsic tyrosine kinase domain in the cytoplasmic tail. The classical EGF receptor is also known as HER1 (human epidermal growth factor receptor 1) or ErbB1, because of homology with the v-erbB oncogene. All closely related EGF peptides bind ErbB1. The remaining 3 receptors are designated ErbB2 (also referred to as Her2 or the neu protooncogene), ErbB3, and ErbB4. c-ErbB2 is unique in that it does not have an identified ligand. Ligand-induced receptor homo- and heterodimerization is a central feature of signaling by the EGFR. The most plausible model for receptor dimerization proposes 2 receptor-binding sites on each monomeric EGF-related