Mutant KRAS in aberrant crypt foci (ACF): Initiation of colorectal cancer?

Biochimica et Biophysica Acta 1756 (2005) 83 – 96 http://www.elsevier.com/locate/bba

Review

Mutant KRAS in aberrant crypt foci (ACF): Initiation of colorectal cancer? Theresa P. Pretlow*, Thomas G. Pretlow Department of Pathology, Case Western Reserve University, Cleveland, OH 44106, USA Received 6 May 2005; received in revised form 22 June 2005; accepted 23 June 2005 Available online 18 July 2005

Abstract Since aberrant crypt foci (ACF) were first described in 1987, they have been the subjects of hundreds of papers; however, the debate continues about their role in colorectal tumorigenesis. This review focuses on the many phenotypic, genetic and epigenetic alterations in ACF that support the hypothesis that ACF are putative precursors of colorectal cancer in both humans and experimental animals. Human ACF, both with and without dysplasia, are monoclonal and display evidence of chromosomal instability. Both of these characteristics are shared by colorectal cancers. While most ACF do not have APC mutations, a large proportion has KRAS mutations and methylated SFRP1 and SFRP2 genes. This epigenetic inactivation gives rise to constitutive Wnt signaling in these putative precursors of colorectal cancer. D 2005 Elsevier B.V. All rights reserved. Keywords: KRAS; Colorectal cancer; Aberrant crypt foci; Premalignant lesion; Putative precursors of colorectal cancer

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . How are ACF defined? . . . . . . . . . . . . . . . . . . . . . . . . ACF in experimental animals . . . . . . . . . . . . . . . . . . . . . 3.1. The biology of ACF in rodents . . . . . . . . . . . . . . . . 3.2. Phenotypic alterations of ACF in rodents . . . . . . . . . . . 3.3. Genetic alterations of ACF in rodents . . . . . . . . . . . . . 4. ACF in humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. The biology of ACF in humans . . . . . . . . . . . . . . . . 4.2. Phenotypic alterations of ACF in humans . . . . . . . . . . . 4.3. Genetic and epigenetic alterations of ACF in humans . . . . . 5. Does mutant KRAS in aberrant crypt foci initiate colorectal cancer? . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Abbreviations: ACF, aberrant crypt foci; PhIP, 2-amino-1-methyl-6phenylimidazo[4,5-b]pyridine; IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; MeIQ, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline; PCNA, proliferating cell nuclear antigen; iNOS, inducible nitric oxide synthase; LOH, loss of heterozygosity; FAP, familial adenomatous polyposis; CEA, carcinoembryonic antigen; hTERT, telomerase protein; CIN, chromosomal instability; MSI, microsatellite instability; HNPCC, hereditary non-polyposis colon cancer; SFRP, secreted frizzle-related protein * Corresponding author. Tel.: +1 216 3688702; fax: +1 216 3681278. E-mail address: [email protected] (T.P. Pretlow). 0304-419X/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbcan.2005.06.002

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1. Introduction This review will focus on aberrant crypt foci (ACF) (Fig. 1), what we have learned about them, and the putative role(s) ACF and/or KRAS play in human colorectal tumorigenesis. We shall cover many aspects of ACF in both experimental animals and humans, but realize that the breadth of the subject does not allow us to be comprehen-

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and will clarify the role of KRAS and ACF in colorectal tumorigenesis.

2. How are ACF defined?

Fig. 1. Aberrant crypt foci (ACF) in methylene blue-stained whole mount of macroscopically normal human colonic mucosa. (A) A focus with less than 20 crypts surrounded by smaller normal crypts with circular lumina. Some of the large crypts appear to have two luminal openings that suggest a recent division of the large crypts. Scale bar = 200 um. (B) A large ACF with elongated, irregular, and/or serrated lumina. Scale bar = 500 um.

sive. For further insights on ACF, we refer the reader to recent reviews on this subject [1 –3]. We will leave the discussion of the many functions and activities of the KRAS oncogene and how these contribute to cancer for the other authors in this issue. The very first description of aberrant crypt foci, or as commonly designated, ACF was in 1987 by Bird [4]. When intact colonic mucosa from carcinogen- (i.e., azoxymethane-) treated mice and rats was fixed, stained with methylene blue and viewed under a microscope, aberrant crypts were observed. They were larger and had a thicker layer of epithelial cells than the surrounding normal crypts [4]. Since then, hundreds of studies have documented the occurrence and biology of these lesions identified microscopically in segments of mucosa from a variety of species including humans [5– 8], hamsters [9,10], and dogs [11]. ACF in all animals, including humans, are heterogeneous in that they contain multiple different genetic, epigenetic, and phenotypic alterations [3,12 – 15] that include mutations in KRAS [16 – 22]. Most studies support the hypothesis that ACF are precursors of colorectal cancer; a few challenge this conclusion. It is hoped that this overview will provide important answers to some of these questions

As originally described [4], ACF are identified and defined by their microscopic appearance in unembedded mucosa. McLellan and Bird [23] defined aberrant crypts as those crypts that (i) are larger than the normal crypts in the field, (ii) have increased pericryptal space that separates them from the normal crypts, (iii) have a thicker layer of epithelial cells that often stains darker, and (iv) generally have oval rather than circular openings. They can be observed as single altered crypts or as a group of altered crypts that appears to form a single unit or focus. ACF (v) frequently are microscopically elevated above the mucosa but also may be depressed, i.e., they usually are not in the same focal plane as the surrounding normal crypts. We consider lesions ACF if they meet four out of five of these criteria in whole-mount preparations of colonic mucosa [20], although it is clear that the evaluation is somewhat subjective and requires experience in identifying these lesions. It is important to emphasize that only lesions that are identified microscopically in the intact mucosa and meet the criteria above are, by definition, ACF. Confusion arises when the criteria of ACF are applied to lesions seen only in histologic sections of colon [24] but not previously verified as ACF in unembedded mucosa. Several investigators have described various early lesions in the colon such as those that accumulate h-catenin [25,26], those that are flat [27], or those that are depleted in mucin [28]. While these newly described lesions may show a stronger association with the development of tumors than the whole spectrum of ACF, there is no convincing evidence that most of these are not subsets of ACF. As discussed here, ACF include a wide range of histologies and biological properties. Classification of these more recently defined lesions as subgroups of ACF allows their development to be investigated from their earliest initiation. A recent paper [29] that describes the differential staining of dysplastic ACF and ACF without dysplasia permits the discrimination of more advanced lesions in the context of their evolution from the population of ACF as a whole. To identify ACF, the mucosa generally is fixed flat to prevent excessive unevenness or curling while viewing; the method and duration of fixation are dictated by the experiments that will be carried out with the ACF. For example, 10% formalin is frequently used when the ACF will only be counted and/or evaluated histologically for the presence of dysplasia. If the ACF are to be immunostained, they need to be fixed according to protocols that preserve the relevant antigens. If DNA and/or RNA are to be extracted from ACF, 70% ethanol for 30 min at 4 -C is a better fixative [30]. Staining the mucosa with 0.2%

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methylene blue allows the crypts to be clearly visualized. Other stains can be used [4,31,32], and some studies have been carried out with unstained mucosa [30]. ACF can also be identified in vivo in humans with high magnification colonoscopy [21,33 – 38]. This will be discussed in. ‘‘The biology of ACF in humans’’.

3. ACF in experimental animals 3.1. The biology of ACF in rodents ACF develop in rats and mice specifically after treatment with colon carcinogens; they do not develop after treatment with carcinogens that do not induce tumors in the colon [39]. These lesions have been observed in rodents as early as 2 weeks after a single dose of the colon carcinogens azoxymethane [23] or 1,2-dimethylhydrazine [40], and their frequency is dose dependent [23,41,42]. ACF are more numerous in the distal colons of rats and mice [23,41,43] where most colon tumors develop [44]. Similarly, the highest frequency of ACF in hamsters is found in the cecum [9] where pathological lesions had been identified previously in this species treated with the same carcinogen [45]. The average number of crypts per focus in rats increases with the number of weeks after a dose of carcinogen [14,40]. At 2 to 3 weeks after carcinogen treatment, most ACF are composed of one or two crypts; at 36 to 41 weeks, approximately 50% of the ACF have four or more crypts [14,40]. This increase in the number of crypts in a focus, and/or the area occupied by the focus as a function of time, suggests that these lesions are growing. Evaluation of isolated crypts from rats treated with dimethylhydrazine demonstrates that aberrant crypts divide by a fission process that begins near the base of the crypt [46]. The varied biological potential or heterogeneity of the ACF induced in carcinogen-treated rats is clearly evident at 36 to 41 weeks: only about half of the ACF have four or more crypts, while the other 50% have stayed the same size or have grown only slightly [14,40]. ACF can also be induced in much smaller numbers in rats by heterocyclic amines such as 2-amino-1-methyl-6phenylimidazo[4,5-b]pyridine (PhIP) [29,47], 2-amino-3methylimidazo[4,5-f]quinoline (IQ) [29,41,48], and 2amino-3,4-dimethylimidazo[4,5-f ]quinoline (MeIQ) [29,41,49]. Since these compounds form when meat and fish are cooked [50], they are likely to be colon carcinogens that we encounter in our diet. While most studies of rodents treated with carcinogens do not describe ACF in the untreated animals, more recent studies report that 42.5 to 57% of 19-week-old F344 rats have small numbers of spontaneous ACF, i.e., ACF that develop in the absence of known exposure to a carcinogen [49,51]. Nearly 23% of the rats had spontaneous ACF with 4 or more crypts [51]. These data suggest a genetic drift or environmental exposure of these F344 rats to a weak carcinogen. It seems unlikely ACF

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were missed in control animals by so many different scientists who have evaluated ACF over the years. ACF induced with MeIQ appeared similar to spontaneous ACF in morphology, immunohistochemically detected proliferating cell nuclear antigen (PCNA), and p53 protein [49]. Spontaneous ACF also have been reported in genetically altered mouse strains that carry a mutant gene for Apc [52] or K-ras [53], but they have not been seen in Apc Min mice without carcinogen-treatment (reviewed in [52]). Increased proliferative activity was measured in rodent ACF by two very different experiments: one evaluated female Sprague – Dawley rats 19 weeks after a single injection with 1,2-dimethylhydrazine by autoradiography of serial cross-sections of crypts labeled with tritiated thymidine [42]; the other used male F344 rats 4 to 36 weeks after a single dose of azoxymethane with immunohistochemical identification of bromodeoxyuridine-labeled cells in longitudinal sections of crypts [54]. While the Sphase cells were increased in ACF compared to their adjacent normal crypts in both studies, neither study found a significant shift of the proliferative zone in the ACF [42,54]. In a more recent study with rats [55], ACF were induced with PhIP, and the proliferating cells were identified with antibodies to Ki-67. Again, the proliferating cells were restricted to the lower portion of the crypts in the majority of the ACF, which, importantly, also were not dysplastic [55]. In 22 of 26 (85%) dysplastic ACF, however, the proliferating cells extended to the upper portions of the crypts [55]. Other studies demonstrated decreased apoptosis in ACF; this resistance to apoptosis in ACF was increased by feeding the rats cholic acid [56]. ACF in rodents have been used extensively as biomarkers to identify substances that enhance or suppress the development of colon cancer [1,57 – 59]. While there are some discrepancies between the ACF assay and the development of colon tumors in these animal assays, the overall agreement is quite high. Early on, it was noted that generally the number of ‘‘large’’ ACF (often defined as 4 or more crypts per focus) [60] or crypt multiplicity (the average number of crypts per focus) [61] demonstrate better association with tumor development than does the total number of ACF. 3.2. Phenotypic alterations of ACF in rodents Dysplasia has long been regarded as an indication of increased risk for progression to cancer. Evaluation of H&Estained sections of ACF from rodents reveals a wide rage of histologies from minor atypia to severe dysplasia [12,40]. Nuclear atypia and/or dysplasia increase as a function of time following a dose of carcinogen; but again there is great heterogeneity [40]. At 41 weeks, some ACF still lack any nuclear atypia [40]. The presence or degree of dysplasia is not associated with the size of the ACF [40]. That at least some ACF have the capacity to progress to cancer is seen in a lesion that was marked with ink in a methylene blue-

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stained, whole-mount preparation that revealed invasive cancer in an H&E-stained section [12]. While the preceding describes ACF induced with 1,2-dimethylhydrazine or azoxymethane, a similar picture was seen in 110 ACF analyzed from rats treated with PhIP [55]. Dysplasia was observed in 30 (27%) of the ACF evaluated, including those from the earliest time point after carcinogen treatment [55]. Dysplastic ACF varied in size from 1 to 16 crypts, with 15 (50%) of these dysplastic ACF having less than 4 crypts [55]. ACF in rats and mice express multiple phenotypic alterations that are also expressed by colorectal cancers induced in these same animals. These alterations include a reduced expression of histochemically demonstrable hexosaminidase and a-naphthyl butyrate esterase activities, increased expression of periodic acid-Schiff reactive material in rats [12,43,62], and increased expression of glutathione-s-transferase isoforms [13]. The most common alteration seen in >99% of ACF induced by azoxymethane in F344 male rats is the marked reduction of hexosaminidase activity [31]. While the significance of this alteration is still unknown, it has been a useful marker to delineate the presence of ACF in rat colon in both unembedded [31] and histological sections [43,54]. Many alterations occur in the same ACF, although none has been observed to be as common as the reduction of hexosaminidase activity [12,43]. Altered expressions of several growth factors and signaling molecules have been observed in rodent ACF, but the alterations do not always parallel those seen in cancers. PKC-hII protein is markedly increased in ACF and tumors, induced with azoxymethane in mice, compared with adjacent normal colonic epithelial cells [63]. Expressions of phosphorylated tyrosine [13], TGFa [13,15,64], and TGFh are markedly reduced in ACF, while expression of EGFR is at a similar level in ACF and adjacent normal epithelium [64]. C-myc and cyclin D1 expressions are increased in ACF of azoxymethane-treated mice [65]. After rats are treated with azoxymethane, the expression of cyclin D1 is increased in both ACF and normal crypts compared to normal crypts in untreated rats [66]; E-cadherin expression is decreased in ACF [66]. Alterations of mucins have long been associated with colon carcinogenesis. Some of these alterations can be detected in ACF in unembedded rat tissue where blue staining sialomucin-producing ACF can be identified in the presence of black sulfamucin-producing normal colonic crypts when stained with high iron diamine-alcian blue (HID-AB) [32]. The production of sialomucin is related to the degree of dysplasia in rat ACF [67]. More recently, mucin-depleted crypts have been demonstrated in wholemount preparations of rat colon [28]. These mucin-depleted crypts appear to be a subset of ACF that are dysplastic and show a better association with the development of colon tumors than the total population of ACF [68]. Aberrant expressions of h-catenin in the nuclei and/or cytoplasm of

colonic epithelial cells of rats have been reported especially in dysplastic ACF [55,65,69,70]. While inducible nitric oxide synthase (iNOS) has been reported in colonic epithelial cells of 100% of dysplastic rat ACF [2], this result is based on the analysis of only three such lesions [69] and therefore needs to be confirmed. Others failed to detect an increase of iNOS in azoxymethane-induced ACF in rats but did detect an increase in tumors [66]. COX-2 expression was seen only in the stromal cells of rat ACF in one study [69]. In a different study, the expression of COX-2 in epithelial cells of rat ACF was not changed compared with epithelial cells in adjacent normal crypts or in crypts from saline-treated rats [66]. 3.3. Genetic alterations of ACF in rodents Mutations in K-ras were the first genetic alterations identified in rodent ACF and tumors induced with azoxymethane [16,17,19]. The incidence of K-ras mutations varied from 7% [17] to 32% [16] in these rat ACF. Similarly, K-ras mutations were identified in 4 of 37 (11%) ACF induced in rats with IQ [48]. Azoxymethane treatment of transgenic mice overexpressing the DNA repair enzyme O 6 -alkylguanine-DNA alkyltransferase resulted in significantly fewer ACF and a smaller proportion of ACF with K-ras mutations than similar treatment of non-transgenic mice [20]. More recently, transgenic mice were developed with targeted expression of mutant K-ras in the epithelial cells of the large and small intestine [53]. Over 80% of the mice over 9 months of age developed tumors spontaneously; most of the tumors were in the small intestines, 7% were in the colon. ACF were observed frequently in the colons of all 5 animals evaluated for ACF [53]. Eleven tumors from these animals were analyzed: none of the 5 informative tumors showed loss of heterozygosity (LOH) for the Apc gene, but 2 of these showed LOH for p53. Six of the non-informative tumors for Apc were stained for h-catenin; all 6 showed only normal membrane staining and lacked nuclear staining indicative of an Apc or hcatenin mutation. These studies clearly demonstrate that mutant Apc is not necessary for the initiation of intestinal cancer and mutant K-ras might be sufficient for initiation, at least in this strain of mice. Colon tumors induced in rats with PhIP or IQ had a higher frequency of b-catenin (Ctnnb1) gene mutations (75%) than Apc mutations (25%) [71], but all 12 colon tumors analyzed had a mutation in one of these two genes. Similarly, ACF induced by PhIP in rats demonstrated Ctnnb1 mutations in 7 of 29 (24%) dysplastic ACF, 6.4% of all ACF evaluated [55], and an Apc mutation in only 1 (3.4%) dysplastic ACF, 0.9% of all ACF [55]. Earlier studies in rats treated with azoxymethane reported Apc mutations in 4 of 28 (13.3%) tumors and in none of 66 ACF [72]. Apc mutations in tumors and/or ACF may be underestimated, however, since the Apc protein is very large and only a small portion of the gene is analyzed. While the rat

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Apc gene exhibits homology with the human APC gene [73], we do not know if the same regions are mutated in rat colon carcinogenesis. The ACF that develop spontaneously in mice that carry a mutant allele of Apc appear to express full-length Apc protein in their ACF [52]. While p53 mutations were detected in 3 of 9 rat colon tumors induced with N-methyl-N-nitrosourea [74], p53 mutations were not detected in tumors induced with azoxymethane or dimethylhydrazine [75 – 77]. Similarly, mutations in p53 were not detected in azoxymethaneinduced mouse colon tumors [78], five microadenomas, and ten microdissected ACF [65]; but all express high levels of p53 protein that is indicative of abnormal p53 protein [65]. The high levels of p53 protein observed in these tumors lack wild-type p53 transcriptional activity [78]. What the role or significance of high levels of p53 protein is in mouse colon tumorigenesis has not been determined for p53 protein that lacks wild-type transcriptional activity but does not appear to be the result of mutation. It is interesting that immunohistochemical detection of p53 protein in rat ACF was one of the earlier alterations reported [79]. In addition to specific gene mutations, genomic instability has been reported in rat ACF by PCR-amplified random polymorphic DNA sequences [80].

4. ACF in humans The first report of ACF in humans was in 1990 [5] followed by more complete descriptions the following year from two different laboratories [6– 8]. Identification of ACF in humans established the validity of the animal models in which these lesions are induced in a short period of time with high doses of carcinogens that are not common in the human environment. More importantly, ACF provide the earliest identified lesions in the colon to investigate the changes that take place during the transformation of normal colonic epithelial cells to colorectal cancer. As will be seen below, many characteristics identified in ACF in rodents are also seen in the ACF from humans with or without colorectal cancer. 4.1. The biology of ACF in humans Most studies to date have been with ACF identified in colons that have been removed surgically from patients for therapeutic reasons. More recently, ACF have been identified in vivo; and some have been removed for analysis [21,33 –38]. No major differences have been reported for ACF obtained with these two approaches. Since human colon is much thicker than rodent colon, the mucosal layer generally is separated from the thick muscle layers before it is viewed under a microscope. Human ACF are variable in size from a single crypt to large areas with over a hundred crypts, and both types of lesions can be found in the same patient (Fig. 1A and B in [6] are from the same patient).

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While many ACF are microscopically elevated, they are macroscopically flat; i.e., even when large, they are not identified as polyps, or lesions, without magnification. In addition, we arbitrarily exclude all lesions that occupy an area of 9 mm2 or greater. It is assumed that these have progressed beyond the ACF stage, even though these lesions are not seen when the colons are examined without a microscope. ACF occur with a higher frequency in the left or distal colon compared to the right or proximal colon, even though the areas of colon examined are usually from patients with colon cancer in those respective areas [6,8,81]. The frequency of ACF is generally higher in patients with colon cancer and/or polyps than in those without [6,34 – 36,38,82,83] and in older compared with younger patients [37,38,82,84]. Also, this age-dependent difference is observed in those without colon cancer [35], and older patients may have larger ACF [38]. The frequency of ACF was increased in patients from geographic areas with a high rate of colon cancer compared with patients from areas with lower rates [85]. Proliferation is increased in human ACF, but like the rodent ACF discussed above, many human ACF do not show a shift in the proliferative zone toward the lumen of the colon [86]. Other studies [81,87,88] do show a shift in the proliferative zone, especially in less frequent ACF with moderate or severe dysplasia. Altered cell and crypt kinetics contribute to the neoplastic process [89] and are part of the basis for the claim that adenomatous ACF originate from hyperplastic ACF [90]. Like rodent ACF, ACF in humans appear to grow by a fission process from the bases of the crypts [91]. This work supports the ‘‘bottom-up’’ hypothesis for the development of human adenomas [92]. A detailed study of ACF detected in vivo in 1998 [35] did much to emphasize the significance of ACF in human colorectal tumorigenesis. These investigators found good associations between the number of ACF, the size of the ACF, or the number of dysplastic ACF detected in the rectal area and the number of adenomas in the rest of the colons of these patients [35]. In a small prospective study, 11 people given sulindac and 9 untreated (controls) were reevaluated for ACF after 8 to 12 months. The sulindac group showed a significant decrease in the number of ACF and complete disappearance in 7 compared with the control group in whom the number of ACF stayed the same or increased slightly [35]. This study nicely demonstrates the utility of this short-term assay to evaluate the effectiveness of dietary changes to alter the development of colorectal cancer in patients that demonstrate a high risk for this disease. The use of a modified colon preparation with a dilute methylene blue enema may facilitate the use of this technique more widely [93]. 4.2. Phenotypic alterations of ACF in humans Dysplasia in the epithelial cells of the colon has long been regarded as a histological marker for increased risk for

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progression to cancer [94]. Consequently, many studies [6,7,21,35,81 – 85,88,90,91,95 –97] have evaluated dysplasia in human ACF. Most agree that the frequency and/or the degree of dysplasia seen in ACF from familial adenomatous polyposis (FAP) patients greatly exceeds that seen in ACF from patients without this syndrome, i.e., those with sporadic colon cancer or without cancer [8,21,88,97]. The frequency of dysplasia in sporadic ACF has been reported to vary from zero [84] or 5 to 10% [8,21,35,85,90,98] to 54% [81]. One reason for the wide variation and/or the low occurrence of dysplasia in most studies is that generally only a single or a few sections of the ACF are evaluated. When serial sections of 50 ACF were cut perpendicular to the luminal openings and evaluated at 100 Am intervals, 15 of 27 dysplastic ACF had less than 50% of the evaluated sections show dysplasia [81]. For example, only 4 of 28, 10 of 54, and 10 of 30 sections evaluated from these ACF showed dysplasia; all ACF classified as dysplastic had at least two sections with dysplasia with 100 Am between them [81]. Other factors that may contribute to the reported differences in the frequency of dysplasia include the study of different populations with different susceptibilities to colon cancer (see [85]) and the study of ACF from different regions of the colon. It is interesting to note that a higher percentage of dysplastic ACF has been reported for the proximal colon (6 of 24 or 25% [85], 2 of 3 or 67% [97], and 11 of 15 or 73% [81]) than for the distal colon (4 of 58 or 7% [85], 3 of 29 or 10% [97], and 15 of 31 or 48% [81]). The grading of dysplasia is subjective, and the criteria used have not always been clearly defined. Also, some have introduced terms like ‘‘heteroplastic ACF (‘‘hetero’’ meaning ‘‘other’’ and ‘‘plasia’’ meaning ‘‘form’’)’’ [88] and ‘‘ACF with mixed type of hyperplasia and dysplasia’’ [83] which may overlap with the ‘‘dysplasia’’ category used by others. This is likely for an ACF illustrated in Pretlow et al. [99] that has a hyperplastic appearance in the luminal portion and dysplasia in the lower portions. While sporadic ACF with severe dysplasia are rare, there is a case report of a 99-yearold patient all of whose ACF that were evaluated had moderate or severe dysplasia [100]. The altered expressions of many proteins in human ACF mirror those seen in colon cancers and polyps and suggest that ACF are precursors of these more advanced lesions. One of the most frequent alterations is the increased expression of carcinoembryonic antigen (CEA) detected in 39 of 42 (93%) ACF immunostained with two monoclonal antibodies [99]. The expression of CEA was not associated with the degree of dysplasia but did increase as a function of size of the ACF. The increased expression and altered location of CEA in human ACF may affect the cell –cell interactions among colonic epithelial cells in ACF [101]. E-cadherin is primarily responsible for cell – cell adhesion in colonic crypts, but P-cadherin may play this role in some circumstances [102]. P-cadherin is not expressed in normal colonic epithelium, but it was expressed in 15 of 23

(65%) human ACF. The expression was independent of dysplasia: 9 of 13 (69%) ACF with atypia (no dysplasia) and 6 of 10 (60%) ACF with dysplasia expressed P-cadherin [102]. All of these ACF continued to have normal Ecadherin expression, a few had cytoplasmic expression of h-catenin, and one had altered gamma-catenin expression [102]. The expression of hexosaminidase and a naphthyl butyrate esterase activities are increased in human ACF compared to adjacent normal mucosa [6]. Note, however, that the alteration of these two enzymes is not as marked or frequent as that observed in the ACF of rodents, and that the alteration is in the opposite direction, i.e., increased rather than decreased [6]. While only a small proportion of ACF showed reduced expression of fragile histidine triad (FHIT) gene, the reduced expression was strongly associated with dysplasia ( P = 0.0002, Fisher’s Exact Test) [103] and may play a role in the progression of lesions in human colon tumorigenesis. Telomerase activity is detected in most human cancers and is postulated to be one of the mechanisms that contribute to the immortality seen in tumors cells [104]. There is a direct relationship between telomerase activity and the expression of telomerase (hTERT) protein [104]. hTERT can be detected at a low level in some normal cells including lymphocytes and at the base of colonic crypts [104]. hTERT showed increased expression in 17 of 45 (38%) ACF; but it appeared to be independent of dysplasia or the number of crypts in the ACF [105]. In these same studies, 11 of 17 (65%) polyps and all 9 carcinomas showed overexpression of hTERT. This increased percentage of lesions, from ACF to cancers, with elevated expression of hTERT (Chi-square, P < 0.01) suggests activation of telomerase activity has a role in the progression of ACF to cancer [105]. The expression of iNOS in human ACF [106] is quite different from that in rodents discussed earlier. By immunohistochemistry, the normal colonic epithelial cells of all patients evaluated had strong cytoplasmic expression of iNOS while 21 of 42 (50%) ACF and 14 of 25 (56%) carcinomas showed a marked reduction of iNOS expression [106]. This reduced expression of iNOS was not related to the degree of dysplasia in ACF or the stage of the cancer. It is interesting to note that iNOS expression in multiple lesions (ACF, polyps, and/or cancer) from the same patient was more similar than expected by random distribution ( P < 0.0001) [106]. Sialyl Lewisx (Lex) and sialyl Tn antigens are overexpressed in >70 to 89% of colon cancers but are not detectable in normal colonic mucosa and are detected only rarely in hyperplastic polyps [107 – 109]. In a study of 54 ACF, 37 (69%) express sialyl Lewisx antigen, 24 (44%) express sialyl Tn antigen, and 20 (37%) express both antigens [14]. Of interest is the fact that the same proportion of ACF express sialyl Lewisx antigen as that seen in adenomatous polyps [109]. Since this ACF study [14] included only 5 ACF from FAP patients and 49 ACF from non-FAP patients, it is unlikely that many of the 37 ACF

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expressing sialyl Lewisx antigen had severe dysplasia. A different conclusion was reached by another laboratory from a study with lectins Dolichus biflorus agglutinin, peanut agglutinin, and monoclonal antibody CA 19-9; the staining properties of the ACF from FAP patients resembled adenomatous polyps while ACF from patients without FAP resembled hyperplastic polyps [88]. Overexpression of glutathione-S-transferase P1-1 (GSTP1-1) was observed in 16 of 18 (89%) human ACF by immunohistochemistry [110]. All of these GSTP1-1 positive ACF also stained for p21K-ras while the remaining two ACF were negative for both GSTP1-1 and p21K-ras [110]. The overexpression of GSTP1-1 appears to be induced by mutant KRAS [110]. In a later study, GSTP1-1 was over expressed in 12 of 15 (80%) ACF [111]; COX-2 was not expressed in these same ACF, and apoptosis (evaluated by the TUNEL assay) was decreased compared to normal mucosa [111]. It appears that GSTP1-1 may protect ACF from apoptosis and thus contribute to the progression of ACF to cancer [111]. The increased expression of p16INK4a correlates inversely with proliferation markers but was seen in 4 of 7 human ACF as well as a high proportion of adenomas and carcinomas [112]. 4.3. Genetic and epigenetic alterations of ACF in humans Human colon cancers are the result of the accumulation of multiple genetic defects in the same cell, i.e., they are monoclonal proliferations [113,114]. If ACF are precursors of these cancers, then ACF also should be monoclonal or neoplastic lesions rather than just hyper-proliferative lesions. A polymorphic site in the first exon of the androgen receptor was used to investigate X-inactivation patterns in human ACF [115]. This study of 11 ACF included: 4 without dysplasia, i.e., displayed only atypia, and 7 with mild dysplasia; 5 from the distal colon, 1 from the transverse colon, and 5 from the proximal colon. Non-random Xinactivation that was not seen in the normal crypts from the same patients was observed in all 11 ACF [115]. This suggests that, from their earliest development, human ACF are monoclonal expansions and are the earliest identified neoplastic lesions in the colon. KRAS mutations in 11 of 15 (73%) ACF, and not in 27 samples of microscopically normal mucosa from the same patients, were the first genetic alterations to be identified in human ACF [18]. Numerous studies from many countries (Canada [116], Italy [117], Japan [21,84,90], and the United States [97,98,118]) have confirmed the presence of KRAS mutations in human ACF, although the frequency has varied widely from 16 of 125 (13%) [116] to 19 of 20 (95%) ACF [98]. The wide variation in the frequency of KRAS mutations reported is likely due to many factors. ACF from FAP [21,116] and ulcerative colitis [116] patients rarely have KRAS mutations. Other genetic backgrounds, different diets and/or life-styles, and different exposures to carcinogens may also influence both the number and genetic alterations of ACF reported in various studies. It is

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interesting to note that tobacco use is associated with increased numbers of ACF [37] as well as increased risk for adenomatous polyps [119], hyperplastic polyps [119], and colon cancer [120]. While the ACF from smokers have not been analyzed for KRAS mutations, benzo[a]pyrene, present at 20 to 40 ng/cigarette, is known to induce a high percentage of G Y T transversions in the lung [121]. In our small study [18], we found 9 out of the 11 ACF with KRAS mutations had G Y T transversions. In addition to these variables within the patient populations, the various laboratories have used different techniques with different sensitivities to identify KRAS mutations in human ACF. The identification of different KRAS mutations (i) in different ACF from the same patient [18,117] and (ii) in ACF and cancer from the same patient [84] suggest ACF as well as tumors occur independently and do not develop from a previous KRAS mutation that involves a large area of colonic mucosa. Mutations of the APC gene are known to occur in polyps, the benign precursors of most colorectal cancers [122], and have been proposed to be the gatekeeper for colon tumorigenesis [123]. In contrast to KRAS mutations, APC mutations are much more difficult to identify since the mutations are spread over a very large protein. Numerous reports have found only a low frequency (3 of 65 (4.6%) [116], 1 of 20 (5%) [98], 5 of 84 (6%) [124], and 0 of 36 [21]) of somatic APC mutations in human ACF from nonFAP patients. Mutations in p53 are thought to be late events in colon carcinogenesis [123,125]. The failure to detect the expression of p53 protein in 57 ACF by immunohistochemistry [84], p53 mutations in 0 of 14 ACF [117], and p53 mutations in 1 of 23 ACF [118] is, therefore, not surprising. Why the overexpression of p53 is detected in rat and mouse ACF, in the absence of p53 mutations, but not in human ACF is likely due to species differences and/or different antibody staining procedures in different laboratories. Recent studies found BRAF mutations in 2% of 53 ACF, and in 43% of hyperplastic polyps, 75% of serrated adenomas, and 33% of mixed polyps from patients with multiple/large hyperplastic polyps and/or hyperplastic polyposis [126]. These results suggest either that ACF are not precursors of serrated adenomas or that the BRAF mutation occurs at a later phase than ACF. Several additional histochemical studies with human ACF demonstrate the expressions of oncogenes and tumor suppressor genes that are known to play a role in colon tumorigenesis. h-catenin is normally highly expressed in the cell membrane and not detected in the cytoplasm or nucleus of colonic epithelial cells [127]. In ACF with dysplasia, 25 of 46 (54%) had cytoplasmic expression; a smaller percentage of these had, in addition, reduced membrane expression and/or increased nuclear expression of h-catenin [127]. The cytoplasmic expression of h-catenin increased from 41% of the 34 ACF with mild dysplasia to 86% of the 7 ACF with moderate dysplasia to 100% of the 5 ACF with severe dysplasia ( P = 0.0003, Poisson loglinear model) [127]. Among the 48 ACF with atypia but no

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dysplasia, only 4% had cytoplasmic expression of h-catenin [127]. These data demonstrate a clear difference between ACF classified with atypia and those with dysplasia. Twenty-five ACF without dysplasia and 18 with dysplasia that had been evaluated for h-catenin expression, were also evaluated for c-myc, a target of activated h-catenin [128]. Increased c-myc expression was seen in 15 of 43 (34.9%) ACF and in 10 of 18 (55.6%) dysplastic ACF [128]. While concordant expression of c-myc and h-catenin was seen in 70% of these ACF, 8 ACF (19%) had increased c-myc expression in the absence of altered h-catenin expression [128]. The increased expression of c-myc in ACF suggests an expansion of the immature colonocytes that normally express c-myc at higher levels than their mature counterparts [129]. Aberrant expression of h-catenin can be the result of a mutation in h-catenin, which is rare in humans, or the altered expression of one of the proteins with which it interacts such as Apc or E-cadherin. Only 4 of 12 (33%) ACF with altered h-catenin expression had reduced Apc expression; all 14 ACF with normal h-catenin expression had normal Apc expression [128]. These studies corroborate the low frequency of APC mutations that have been reported in human ACF and are discussed elsewhere in this review. The low frequency of reduced Apc expression in ACF also suggests that the APC gene is not frequently silenced by methylation [130] at the ACF stage although this has not been tested directly. Of the 46 ACF previously evaluated for h-catenin, only 3 ACF with severe dysplasia and 1 of 3 ACF with moderate dysplasia showed reduced expression of E-cadherin (Hao and Pretlow, unpublished data). These studies suggest that the expression of the proteins c-myc and h-catenin are altered more frequently than Apc or Ecadherin in human ACF. It appears that c-myc and hcatenin play roles early in colon tumorigenesis that in some cases are independent of Apc and might reflect activation of the Wnt pathway by methylation of secreted frizzled-related protein genes (SFRPs) that is seen frequently in ACF [131]. Most human colon cancers are aneuploid with multiple chromosomal aberrations [132]. This has given rise to the hypothesis that most human colorectal cancers display chromosomal instability (CIN) [133], but questions remain as to how early CIN is observed and whether this is the cause or the result of chromosomal aberrations? Fingerprints of 44 ACF, 23 cancers, and normal crypts from the same patients generated by random primers with PCR, were compared. Altered fingerprints, that suggest CIN, were observed in 23.3% of the ACF and 95.7% of the cancers [134]. More recently, microdissected epithelial cells from 32 ACF were analyzed with 8 microsatellite markers. A total of 8 allelic alterations including 5 LOH and 3 microsatellite instability (MSI) were seen in 7 (22%) different ACF [135]. The LOH and altered fingerprints observed in these ACF suggest that CIN occurs very early and might be the origin of aneuploidy seen later in many colorectal cancers. Since over 90% of ACF lack APC mutations, the finding of LOH

in ACF suggests CIN can occur before APC mutations. Patients with hereditary non-polyposis colon cancer (HNPCC) and about 15% of those with sporadic colon cancer have cancers with defective DNA mismatch repair enzymes [136,137]. This results in MSI in these tumors. MSI has been demonstrated with a similar frequency in ACF [138,139]. The presence of MSI in these very early lesions suggests ACF with MSI contribute to the development of cancers with this phenotype and places these ACF as logical precursors of MSI cancers. In addition to these genetic alterations that have been documented in ACF, there are several studies that implicate a role for DNA methylation very early in colorectal tumorigenesis. The first study to demonstrate methylation in ACF reported 21 of 61 (34%) ACF with methylation of p16, MGMT, hMLH1, MINT31, MINT2, and/or MINT1 [97]. Methylation was less frequent in ACF from FAP patients (3 of 27 or 11%) compared to that in ACF from non-FAP patients (18 of 34 or 53%) [97]. The methylation of the individual loci varied from 3% for hMLH1 to 21% for MINT31 [97]. A high frequency of methylation in human ACF was found for the newly described SLC5A8 gene, a sodium transporter that is implicated in colon cancer [140]. In other studies, 19 of 35 (54%) ACF and 15 of 22 (68%) cancers showed hypermethylated of the cellular retinolbinding protein 1 (CRBP1), MINT31, or H-cadherin (CDH13) [141]. ACF and/or cancers from the same patients did not show similar methylation patterns for these genes [141]. One of the most interesting findings is the frequent methylation of the secreted frizzle-related protein (SFRP) genes in ACF [131]. Analysis of 15 ACF found SFRP1 methylated in 14 and SFRP2 methylated in 13 ACF. This epigenetic inactivation allows constitutive Wnt signaling in these putative colon cancer precursors that usually lack APC mutations. The major alterations observed in human ACF are summarized in Table 1.

5. Does mutant KRAS in aberrant crypt foci initiate colorectal cancer? From the discussions above, there are multiple reasons to conclude that ACF are the earliest precursors of colorectal cancer identified to date. Most agree that ACF with advanced or severe dysplasia, the few that have been identified with APC mutations, are precursors of colorectal cancers [98]. In fact, Kinzler and Vogelstein [123] placed the ‘‘dysplastic ACF’’ immediately following the APC mutation but before the KRAS mutation in their diagrammatic representation of proposed changes that take place between a normal colonic epithelial cell and the ultimate colorectal cancer. But is the severely dysplastic ACF and/or the ACF with an APC mutation the only ACF that is a legitimate precursor of colorectal cancer? Many of the phenotypic, epigenetic, and genetic alterations in both

T.P. Pretlow, T.G. Pretlow / Biochimica et Biophysica Acta 1756 (2005) 83 – 96 Table 1 Molecular alterations in human ACF Molecular alteration

Frequency (%)

References

APC mutations h-catenin expression BRAF mutations CEA expression CIN c-myc expression Dysplasia

0–6 54 2 93 16 – 23 35 0 – 54

GSTP-1 HTERT expression INOS expression KRAS mutations MSI Methylation of CDH13 Methylation of CRBP1 Methylation of hMLH1 Methylation of MGMT Methylation of MINT1 Methylation of MINT2 Methylation of MINT31 Methylation of p16 Methylation of SFRP1 Methylation of SFRP2 Methylation of SFRP4 Methylation of SFRP5 Methylation of SLC5A8 P-cadherin expression Sialyl Lewisx expression Sialyl Tn antigen expression

80 – 89 38 50 13 – 95 10 14 37 3 12 8 5 11 – 21 4 93 87 40 53 47 65 69 44

[21,98,116,124] [127] [126] [99] [134,135] [128] [6 – 8,21,35,81 – 85, 88,90,91,95 – 98] [110,111] [105] [106] [18,21,84,90,97,98,116 – 118] [138,139] [141] [141] [97] [97] [97] [97] [97,141] [97] [131] [131] [131] [131] [140] [102] [14] [14]

animal and human ACF mirror those seen in colorectal cancers. These alterations occur at a much higher frequency than the 5% of ACF detected with APC mutations. Even among the colorectal cancers that display chromosomal instability (CIN) [133], not all have APC mutations. While several genes such as APC, KRAS, and p53 are mutated at a high frequency (>50%) in colorectal cancers [142], multiple other genes are also altered in these cancers [132]. More recently, the analysis of 106 colorectal tumors demonstrated that 44% of these tumors had only one of these three genes mutated, only seven (6%) had all three genes mutated, and twelve (11.3%) lacked mutations in any of these three genes [143]. These and many other studies affirm that many additional genes, besides those that have been characterized to date, are involved in colon tumorigenesis. While multiple genes may be involved in colorectal tumorigenesis, what is the evidence that APC mutations are the ‘‘gatekeeper’’ [123] or initiator of this process? Many studies affirm that APC mutations occur early during the development of a high percentage of colorectal cancers [21,122,142]. The placing of APC mutation before KRAS rests largely on the low frequency (13%) of KRAS mutations in 40 tumors that ‘‘were generally small tubular adenomas with low-grade dysplasia.... from seven patients with familial adenomatous polyposis [FAP]’’ [142]. More recent studies of small adenomas from patients with sporadic

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colorectal cancer have found as high a frequency of KRAS mutations in small adenomas as in large ones [21,144]. A detailed study of ACF and adenomas from patients with and without FAP suggests that early colorectal tumorigenesis may be different in these two settings [21]. In non-FAP patients, APC mutations were lacking in 100% of ACF; but KRAS mutations occurred in 17 of 27 (63%) ACF with dysplasia and 89 of 106 (82%) ACF without dysplasia. Polyps from these same non-FAP patients had APC mutations in 24 of 31 (78%) and KRAS mutations in 20 (65%), the same percentage as in dysplastic ACF. In FAP patients, dysplastic ACF had somatic APC mutations in 100% but KRAS mutations in only 1 of 8 (13%). All of the polyps from FAP patients had somatic APC mutations, and 78% had KRAS mutations [21]. Thus, it appears that KRAS mutations precede APC mutations in non-FAP patients, while APC inactivation precedes KRAS mutations in FAP patients. In addition, recent studies of a transgenic mouse with targeted expression of mutant K-ras that developed ACF and tumors without Apc or h-catenin mutations demonstrate that colorectal tumorigenesis can be initiated in the absence of an Apc or b-catenin mutation [53]. Can we now say that KRAS mutations in ACF initiate sporadic colorectal cancer? Since all ACF do not appear to have KRAS mutations, we might argue that there are multiple ‘‘initiators’’ of colorectal cancer: APC mutations for FAP tumors, KRAS for most sporadic tumors that have KRAS mutations, and one or more unknown genes for sporadic ACF and tumors lacking KRAS mutations. Alternatively, there might be one or more ‘‘initiators’’ of sporadic ACF that subsequently acquire a KRAS mutation. Some ACF with or without KRAS mutations could be immediate precursors of hyperplastic polyps since hyperplastic polyps also possess a high frequency of KRAS mutations [145]. This does not necessarily remove these ACF from the path to cancer since cancer has been described in some hyperplastic polyps [146,147]. There is also the intriguing finding that the Wnt pathway is epigenetically activated at a high frequency in ACF by hypermethylation of the SFRP1 and SFRP2 genes [131]. It is thought that the activated Wnt pathway provides the growth advantage for these clones of cells until the appropriate mutations can occur. While these ACF are unlikely to have APC mutations, there is no information regarding when these genes are methylated in respect to the acquisition of KRAS mutations. As noted in an editorial recently, ‘‘it is still controversial in which step the K-ras mutation occurs’’ [148]. It appears increasingly likely there are multiple paths with different starting points or initiators leading to the disease(s) we call colorectal cancer.

Acknowledgements This work was partially supported by NIH grants CA66725 and CA43703. The authors thank Dr. Leonard Augenlicht for his insightful comments and Jessica Laux for

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the photomicrographs. We wish to express our deep appreciation and gratitude to our many collaborators, research assistants, students, and research associates who have contributed to this work over many years.

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