Relationship of gastrin processing to colon cancer

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GASTROENTEROLOGYVol. 109, No. 4

Relationship of Gastrin Processing to Colon Cancer See article on page 1 1 4 2 .

esearch in gastrointestinal peptides has traditionally related their secretion to specific physiological effects such as gastric acid or pancreatic secretion. With a better understanding of cell biology, recent investigations have focused on the role that peptides play in the growth and differentiation of the gut. Thus, in addition to its well-known regulation of gastric acid secretion, gastrin has been ascribed to have a proliferative action on normal and malignant gastrointestinal tissues. The role of gastrin in the proliferation of colon cancers has been of considerable interest for many years, and an excellent review of the topic recently appeared in GASTROENTEROLOGY.1 What then could be new in this field? The answer to that question relies on the fact that gastrin and most other peptide hormones require extensive posttranslational processing for full biological activity. 2 The report by Ciccotosto et al. in this issue 3 is an important chapter in a developing story that progastrin processing intermediates may have distinct effects on the growth and differentiation of the gut. As is the case with other gut peptides, gastrin's primary translation product, progastrin, undergoes extensive posttranslational processing in G cells of the gastric antrum. Ciccotosto et al. suspected that gastrin processing might not be as efficient in tumor ceils and thus measured unprocessed and fully processed gastrins in human colorectal carcinomas with four different gastrin antisera. They noted that only 69% of the tumors contained fully processed gastrins but that 100% had detectable amounts of progastrin. In addition, they correctly surmised that progastrins may not be stored in the tumors but rather secreted from these tumor cells into the extracellular milieu. As might be anticipated, plasma levels of total but not processed gastrins were increased in patients with colorectal carcinomas. This was true for patients with colon cancer with and without Hdicobacterpylori infection (a known cause of hypergastrinemia). The authors concluded that unprocessed gastrins may be important autocrine growth factors in human colorectal cancer.

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Gastrin Posttranslational Processing

To understand the potential trophic and other biological effects of various molecular forms of gastrin, it is necessary to review gastrin's posttranslational processing. Gastrin serves as an ideal model of prohormone

processing mechanisms because many of its processing reactions are common to other prohormones. The structure of human preprogastrin consists of a signal peptide as well as amino- and carboxy-terminal extensions flanking gastrin tetratriacontapeptide (G34) (Figure 1). Peptide hormones and other secreted proteins are initially synthesized on ribosomes from the amino-terminal end and enter the cell's secretory pathway via translocation into the endoplasmic reticulum. The "pre" or signal peptide of preprogastrin facilitates this translocation, but it is cleaved from the propeptide in the endoplasmic reticulum and not secreted under normal circumstances. 4 The newly synthesized polypeptide chain (progastrin) then proceeds from the endoplasmic reticulum to the Golgi stack, where it can undergo further posttranslational modification. Progastrin undergoes tyrosine sulration to a variable extent in the Golgi. Whereas sulfation is essential for the biological action of cholecystokinin (CCK) at CCKa receptors located in the gallbladder and pancreas, the acid secretagogue effect of gastrin at gastrin/CCK~ receptors is not influenced by the presence or absence of tyrosine sulfation) '6 Proteins exiting cells via either the constitutive or regulated secretory pathways share a common trail from the endoplasmic reticulum through the Golgi stack but diverge in the trans-Golgi network, where proteins are sorted according to their final destination] Proteins in the constitutive pathway generally do not undergo extensive posttranslational processing (e.g., albumin secretion from hepatocytes) and are transported in secretory vesicles that continuously fuse with the plasma membrane. Conversely, polypeptide hormones synthesized in neuroendocrine cells enter the regulated pathway, are stored, and are processed in secretory granules for hours or days before secretagogue-induced release, as can be seen with gastrin produced in antral G cells. After exiting the trans-Golgi network, progastrin is concentrated in secretory granules containing several processing enzymes (Figure 1) that are shared with other neuroendocrine cells. The tissue-specific expression of these enzymes is important because the nature of the resultant products is dependent on the presence of the enzymes. 8 Progastrin's amino- and carboxy-terminal extensions are removed by a prohormone convertase with cleavage at arginine/arginine dibasic residues. The carboxy-terminal basic amino acids are then sequentially removed by carboxypeptidase H, which results in the formation of glycine-extended G34. Conversion of the

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EDITORIALS 1385

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Figure 1. Gastrin posttranslational processing. Preprogastrin is synthesized on the endoplasmic reticulum (E.R.), and its "pre" or signal peptide is cleaved just after translocation into the endoplasmic reticulum lumen. Progastrin and other secretory proteins are then transported to the Golgi, where they are often sulfated at a tyrosine residue (Tyr). In well-differentiated neuroendocrine cells, progastrin is sorted in the transGolgi network to the regulated pathway of secretion and into secretory granules that contain the enzymes necessary to complete progastrin processing. In these granules, the amino- and carboxy-terminal flanking regions are cleaved by a prohormone convertase at dibasic arginine residues (ArgArg) and the carboxy-terminal arginines removed by carboxypeptidase H revealing G34-Gly. G34-Gly can then be amidated by PAM to form G34-NH2 or cleaved at internal lysine/lysine (LysLys) residues, resulting in the production of G17-Gly. Recent biosynthetic studies 9 suggest that although G34-NH2 can be cleaved at the LysLys site by a prohormone convertase to yield G17-NH2, G17-Gly is not amidated by PAM. Thus, G17-Gly may be a distinct end product of progastrin processing in antral G cells. In general, only amidated and glycine-extended forms of gastrin are produced in neuroendocrine cells. Conversely, gastrin in poorly differentiated nonendocrine cells ~ found in colon cancers bypasses the processing machinery found in secretory granules and is secreted in larger, unprocessed forms that exit ceils via the constitutive secretory pathway.

Gly-extended peptide to a peptide amide via the action of peptidylglycine 0~-amidating monooxygenase (PAM) and cleavage of a lysine/lysine pair via another prohormone convertase completes the conversion of progastrin to amidated gastrin heptadecapeptide (G17-NH2).2 Previously, it was believed that the order of these last two steps in progastrin processing was irrelevant to the formation of the final product. However, recent biosynthetic studies show that the majority of G17-NH2 arises from the conversion of the G34-NH2 to G17-NH2 rather than by amidation ofG17-Gly. 9 This suggests that the conversion of G17-GIy to amidated G17 is blocked and that G17-Gly is a second distinct end product in progastrin processing.

Quantification of Gastrins Given all of the different molecular forms of gastrin, it is easy to see that no single antiserum will detect every form of gastrin. This fact often accounts for the reported differences in gastrin content found in colon cancers. The term "gastrin" usually refers to G17-NH2, because this is the predominant form of gastrin in the gastric antrum. Consequently, most gastrin antisera are specific for the carboxy-terminal amide residues of G17NH2 and thus do not detect glycine-extended or other forms of unprocessed gastrin. This is of particular concern when examining gastrins in colon cancers, because dedifferentiated tissues often lose the ability to completely

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process prohormones, m Moreover, cells without a welldefined regulated pathway of secretion, such as colon carcinomas, do not efficiently process progastrin in the constitutive pathway and thus produce mostly unprocessed gastrins, ll To ensure detection of all forms of processed and unprocessed gastrin, Ciccotosto et al. used an assay that recognizes the carboxy-terminal extension of progastrin. This progastrin antiserum should detect the carboxy-terminal extension both after intracellular cleavage with eventual formation of G17-NH2 and when it remains attached to larger unprocessed gastrins. Using this assay, the authors detected increased amounts of progastrin in many of the tumors that would have been missed with a standard gastrin radioimmunoassay that is specific for the carboxy terminus of G17-NH2. These results are consistent with previously published data using similar methods) 2'13 Because small peptide fragments, such as the carboxy-terminal extension, are often rapidly degraded in plasma, the authors used a processing independent gastrin assay to quantify total gastrin. For this assay, they treated colon cancer extracts and plasma with trypsin to completely cleave any larger, unprocessed gastrins at the lysine/lysine site. All cleaved gastrins (total gastrin) were then detected with antisera specific for the amino terminus of G17. With this assay, they found elevated plasma levels of total gastrin in patients with colorectal cancer.

Relationship of Gastrin Processing to Physiology Although this is an interesting observation to peptide chemists, it was thought to be of little relevance to cancer or to the biology of the gut because complete posttranslational processing with carboxy-amidation of G17 and G34 is required for binding to gastrin/CCKB receptors) 4 More importantly, the carboxy-terminal amide moiety is necessary to stimulate acid secretion from parietal cells, 15 and amidated gastrins are used in gastrin proliferation studies.16'17 Thus, glycine-extended gastrins (G-Gly) and other progastrins were believed to have no physiological importance other than to serve as precursors for the synthesis of amidated gastrin. Confirmation of this hypothesis was found in the fact that G-Gly was at least four orders of magnitude less potent than amidated G17-NH2 in acutely stimulating gastric acid secretion) 5,18 Nevertheless, interest in the physiological effects of G-Gly has been fueled by the observations that G-Gly is stored in brain ~9 and gut tissues, 2°'2i secreted with G-NH222 from antral G-cells into the circulation, and achieves concentrations in plasma roughly equivalent to those of G-NH2 .23'24 Moreover, G-Gly is found in greater

GASTROENTEROLOGY Vol. 109, No. 4

concentrations than G-NH2 during development and in some malignant tissues that express gastrin, such as Zoll i n g e r - E l l i s o n tumors. 25'26 Thus, the evidence pointed to G-Gly's role as a growth factor but not as a direct acid secretagogue. Therefore, to explore this possibility and to link gastrin processing to gastrointestinal physiology, we noted that both G17-NH2 and G17-Gly stimulated DNA synthesis in a dose-dependent fashion in the exocrine pancreatic AR4-2J cell l i n e s The stimulation induced by G17-NH2 was completely reversed by two different selective gastrin/CCKB receptor antagonists, whereas neither antagonist decreased growth induced by G17-Gly. These data suggested that G17-Gly might act through a mechanism independent of gastrin/CCKt3 receptors. Indeed, competitive binding studies suggested the presence of a unique receptor for G17-GlyS Others have shown that pancreatic AR4-2J cells secrete G-Gly, which stimulates growth in an autocrine fashion as hypothesized for colon cancers in v i v o . 28 In this model, synthesized progastrin would be only partially processed in tumor cells within the constitutive pathway of secretion. Thus, only small amounts of gastrin products would be stored within the cell, and mostly unprocessed gastrins would quickly exit the cell to stimulate growth. If this model is correct for G-Gly, how can one rationalize the fact Ciccotosto et al. did not find higher plasma G-Gly concentrations in patients with colon cancer? Two explanations seem plausible. First, the amounts of G-GIy in plasma are at the lower limit of detection for the assay; thus, the small changes may not be easily shown. Second, the dissociation constant for G17-Gly binding and its 50% effective concentration for growth stimulation are both quite low at 10 -l° mol/L.27 G-Gly could easily function as an autocrine growth factor at these concentrations with minimal cellular secretion, which would result in a negligible increase in plasma GGly. Finally, because Ciccotosto et al. did find elevated plasma levels of total gastrin in patients with colorectal cancer, it is possible that there are other receptors for as yet unidentified growth-promoting progastrin processing intermediates. Other data help to confirm the role that G-Gly may play in gastrointestinal physiology. Although acute administration of G-GIy has no effect on gastric acid secretion, ~5'1s long-term administration of G-Gly markedly enhances stimulated but not basal acid secretion from isolated parietal cells and in vivo via an increase in the expression of H+,K ÷adenosine triphosphatase within gastric parietal cells. 29'3° If gastrin/CCKB and G-Gly receptors are distinct, one might expect that they would have distinct signal transduction mechanisms. Binding of G17-NH2 to its receptor stimulates the mobilization of intracellular Ca2+, whereas G17-

October 1995

Gly binding does not seem to alter intraceilular Ca 2+ or adenosine 3',5'-cyclic monophosphate concentrations. 29 Although tyrosine kinase inhibitors block the effects of G17Gly on parietal ceils, the exact nature of its signal transduction awaits the isolation of a complementary DNA encoding the receptor and further study. Singh et al. have identified a receptor distinct from CCKa and gastrin/CCKB receptors that mediates the proliferative effects of G-NH2 and GGly. 31 This receptor seems to be different from the G-Gly receptor in that it recognizes G-NH2 and G-Gly with equal affinity and requires much higher concentrations of the peptides to mediate its trophic actions. Other fruitful areas of investigation in the field will likely include determination of whether G17-NH2 and G17-Gly act independently or cooperatively to stimulate growth and precise identification of the enzymes responsible for the generation of these and other products of progastrin posttranslational processing. Taken as a whole, these observations suggest that growth-related receptors for G-Gly may mediate physiological or pathophysiological effects of the products of progastrin processing. Furthermore, this suggests for the first time that the precursor and the product of a key posttranslational processing reaction, peptide O~-amidation, have distinct biological actions mediated through separate receptors. Indeed, the posttranslational processing of a prohormone may define the ultimate physiological effects of its products. Because other peptide hormone processing intermediates are also found in high concentrations in tumors and during development, we anticipate that these intermediates may also have physiological effects that will be defined by future studies. CHRIS J. DICKINSON Department of Pediatrics University of Michigan Medical Center Ann Arbor, Michigan

References 1. Rehfeld JF. Gastrin and colorectal cancer: a never-ending dispute? Gastroenterology 1995; 108:1307-1309. 2. Merchant JL, Dickinson CJ, Yamada T. Molecular biology of the gut: model of gastrointestinal hormones. In: Johnson LR, ed. Physiology of the gastrointestinal tract. 3rd ed. New York: Raven, 1994:295-350. 3. Ciccotosto GD, McLeish A, Hardy K_I, Shulkes A. Expression, processing, and secretion of gastrin in patients with colorectal carcinoma. Gastroenterology 1995; 109:1142-1153. 4. Walter P, Gilmore R, Blobel G. Protein translocation across the endoplasmic reticulum. Cell 1984;38:5-8. 5. Chowdhury JR, Berkowitz JM, Praissman M, Fara JW. Effect of sulfated and non-sulfated gastrin and octapeptide-cholecystokinin on cat gall bladder in vitro. Experientia 1976;32:11731175. 6. Gregory RA, Tracy HJ. The constitution and properties of two gastrins extracted from hog antral mucosa. Gut 1964;5:103114. 7. Rothman JE, Orci L. Molecular dissection of the secretory pathway. Nature 1992; 355:409-415.

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8. Steiner DF, Smeekens SP, Ohagi S, Chan SJ. The new enzymology of precursor processing endoproteases. J Biol Chem 1992; 267:23435- 23438. 9. Varro A, Voronina S, Dockray GJ. Pathways of processing of the gastrin precursor in rat antral mucosa. J Clin Invest 1995;95: 1642-1649. 10. Rehfeld JF, Bardram L, Blanke S, Bundgaard, JR, Friis-Hansen L, Hilsted L, Johnsen AH, Kofod M, Luttichau HR, Monstein HJ, Nielsen C, Nielsen FC, Paleheimo LI, Pedersen K, Pildal J, Ramlau J, van Solinge WW, Thorup U, Odum U. Peptide hormone processing in tumours: biogenetic and diagnostic implications. Tumour Biol 1993;14:174-183. 11. Dickinson CJ, Takeuchi T, Guo Y-J, Stadler BT, Yamada T. Expression and processing of prohormones in nonendocrine ceils. Am J Physiol 1993;264:G553-G560. 12. Kochman ML, DelValle J, Dickinson CJ, Boland CR. Post-translational processing of gastrin in neoplastic human colonic tissues. Biochem Biophys Res Commun 1992; 189:1165-1169. 13. van Solinge WW, Neilsen FC, Friis-Hansen L, Falkmer U, Rehfeld JF. Expression but incomplete processing of progastrin in colerectal carcinomas. Gastroenterology 1993; 104:1099-1107. 14. Kopin AS. Lee YM, McBride EW, Miller LJ, Lu M, Lin HY, Kolakowski LF, Beinborn M. Expression cloning and characterization of the canine parietal cell gastrin receptor. Proc Natl Acad Sci USA 1992; 89:3605-3609. 15. Matsumoto M, Park J, Sugano K, Yamada T. Biological activity of progastrin posttranslational processing intermediates. Am J Physiol 1987;87:G315-G319. 16. Johnson LR, McCormack SA, Wang J-Y: Regulation of gastrointestinal mucosal growth. In: Walsh J, ed. Gastrin. New York: Raven, 1993:285-300. 17. Smith JP, Solomon TE. Effects of gastrin, proglumide, and somatostatin on growth of human colon cancer. Gastroenterology 1988; 95:1541-1548. 18. Hilsted L, Hint K, Christiansen J, Rehfeld JF. Neither glycineextended gastrin nor the 1-13 fragment of gastrin 17 influences gastric acid secretion in humans. Gastroenterology 1988;94: 96-110. 19. Rehfeld JF, Hansen HF. Characterization of preprochoiecystokinin products in the porcine cerebral cortex: evidence of different processing pathways. J Biol Chem 1986;261:5832-5840. 20. Sugano K, Aponte GW, Yamada T. identification and characterization of giycine-extended post-translational processing intermediates of progastrin in porcine stomach. J Biol Chem 1985;260: 11724-11729. 21. DelValle J, Sugano K, Yamada T. Progastrin and its glycine-extended posttranslational processing intermediates in human gas~ trointestinal tissues. Gastroenterology 1987;92:1908-1912. 22. Sugano K, Park J, Dobbins WO, Yamada T. Glycine-extended progastrin processing intermediates: accumulation and cosecretion with gastrin. Am J Physiol 1987;253:G502-G507. 23. Hilsted L, Hansen CP. Corelease of amidated and glycine-extended antral gastrin after a meal. Am J Physiol 1988;G665G669. 24. DeIValle J, Sugano K, Yamada T. Gtycine-extended processing intermediates of gastrin and choiecystokinin in human plasma. Gastroenterology 1989;97:1159-1163. 25. Hilsted L, Bardram L, Rehfeld JF. Progastrin maturation during ontogenesis. Accumulation of glycine-extended gastrins in rat antrum at weaning. Biochem J 1988;255:397-402. 26. Pauwels S, Desmond H, Dimaline R, Dockray GJ. Identification of progastrin in gastrinomas, antrum, and duodenum by a novel radioimmunoassay. J Clin Invest 1986;77:376-381. 27. Seva C, Dickinson CJ, Yamada T. Growth promoting effects of glycine-extended progastrin. Science 1994;265:410-412. 28. N6gre F, Fagot-Revurat P, Vaysse N, Rehfeld JF, Pradayrol L. Progastrin induces autocrine/intracrine proliferative effects on

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pancreatic rat tumoral cells (abstr). Gastroenterology 1994; 106: A309. 29. Kaise M, Muraoka A, Seva C, Takeda H, Dickinson CJ, Yamada T. Glycine-extended progastrin intermediates induce H+,K+-ATPase c~-subunit gene expression through a novel receptor. J Biol Chem 1995;270:11155-11160, 30. Higashide S, Gomez G, Townsend CM Jr, Thompson JC, Greeley GH Jr. Glycine-extended gastrin potentiates gastrin-stimulated acid secretion in rats (abstr). Gastroenterology 1995; 108:Al13. 31. Singh P, Owlia A, Espeijo R, Dai B. Novel gastdn receptors medi-

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ate mitogenic effects of gastrin and processing intermediates of gastrin on Swiss 3T3 fibroblasts. J Biol Chem 1995; 270:84298438.

Address requests for reprints to: Chris J. Dickinson, M.D., Department of Pediatrics, University of Michigan Medical Center, 1500 East Medical Center Drive, F6854 Mort, Ann Arbor, Michigan 481090200. Fax: (313) 763-7359. © 1995 by the American GastroenterologicalAssociation 0016-5085/95/$3.00

Colitic Cancer: Supervision, Surveillance, or Surgery? S e e a r t i c l e on p a g e 1 1 8 8 .

ince the introduction of effective medical and surgical treatments in the early 1960s, ulcerative colitis has been associated with a surprisingly low mortality. Severe acute attacks, usually during the first 2 years of disease, are the major killer. Thereafter, there is usually an increased mortality from colorectal cancer, *'2 although the death rate among sufferers from Colitis as a whole differs little from that expected in the general population. ~'3 Some colitic patients have a greater risk of cancer than others. In a series of 3117 patients, the overall risk of colorectal cancer when inflammation extended proximal to the hepatic flexure was approximately 1 in 200 per patient year compared with 1 in 1200 for patients with proctitis and 1 in 660 for patients with disease of intermediate extent. 4

S

How Should Extensive Colitis Be Treated Early in the Disease Course? Colectomy performed early in the disease course removes the colorectal cancer risk. In a recent series reported from a defined geographical area of Copenhagen, where a vigorous policy of medical and surgical treatment is pursued for all patients with colitis, 35 % of patients with total colitis were treated surgically during the first 5 years of disease. 3 Among all 1161 patients, followed up over a median of 11.7 years (range, 0 - 2 6 years), 6 cases of colorectal cancer occurred, corresponding to 6.6 cases expected in the general population. This is the only large study in which no excess cancer risk in colitis has been shown. It seems likely that the cancer risk was affected by the policy of long-term treatment of disease in remission with mesalamine derivatives, ~ rapid medical

treatment of acute attacks with corticosteroids, and early surgical treatment when severe inflammation was unresponsive to medical measures.

How Should Extensive Colitis Be Treated Late in the Disease Course? The paper by Provenzale et al. 6 in this issue of GASTROENTEROLOGY is concerned with the clinical problem of patients with total ulcerative colitis who have had the disease for 10 years and have not been treated surgically for acute or chronic inflammation. Is clinical supervision the best policy, i.e., a follow-up regime during which colonoscopy is performed whenever symptoms suggestive of cancer occur? Should a surveillance program be instituted in which regular endoscopy is performed regardless of symptoms? Or should the patient be advised to undergo prophylactic surgical treatment to avoid the future dangers of a severe acute attack of colitis and colorectal cancer? The computer simulation begins with a notional population of 10,000 such patients, each aged 30 years, whose subsequent survival is calculated based on assumptions about the incidence of lethal colorectal cancer, the death rate after elective or urgent surgical treatment, the fatal complication rate of colonoscopy, and the expected mortality over the years from all causes. The analysis ends when the last patient dies in old age. The authors are to be congratulated not only for considering so many outcome variables but also comparing 17 different options for managing the patients. Sensitivity analyses, in which the value of each parameter is varied over a broad range to show its effect on outcome, identified the incidence of cancer and the annual rate of colectomy for refractory or severe inflammation as critical factors in the choice between supervision,