Pethokgy lniemtionall997; 47: 470-477

Original Article Expression of p21 wAF1’cipl in colorectal adenomas and adenocarcinomas and its correlation with p53 protein expression

WataruYasd,’Yoshihiko Akama,‘ HiroshiYokozaki: Shuho Semba,’Yasusei Kudo,’ Fumio ShimamotoZ and EiichiTaharal ‘First Department of PathologH Hiroshima University School of Medicine and 2Department of Pathology, Hiroshima University Hospital, Hiroshima, Japan

The expresslon of p53-Inducible cydlndependent kinase Inhlbitor, p21mm in non-neopiastlc mucosa, adenoma and adenocarclnoma of the colorectum was examined by immunohlstochemlstry and western Mottlng and I t s relation with the expression of p53 protein was analyzed. Non- neoplastic epithelial cells at the surface area showlng no proltferatlve activity expressed p21WAF1~l.fhe expression of p21w~1m1 was lmmunohlstochemlcally detected in 55% (2OW3m of the adenomas and 66% (1sonSS) of the adenocarclnomas, respectively. The lncldence of strongly posltlve cases was slgnHicantly higher In the adenocarclnomas (27%) than In the adenomas (18%) (fW.05). The incidence of cases wtth strong p2lWAF1- expresslon was higher In stages 0, l and 2 carcinomas than in stages 3 and 4 carcinomas (pcO.05). A decrease in the inc ldem of cases with strong expression was detected in carclnomas Invading deeper than muscularis propria. The lncldence of strongly positive cases was slgniflcantly lower in cadnomas with lymph node metastasis than those without metastasls (pdl.05). The expression of p21 as well as p53 detected by western blotting was compatlble with the results of lmmunohistochemlstry in most cases examined. However, there was no significant correlatlon between the expression of ~ 2 1 ~ - and the abnormal accumulation of p53.These findlngs overall suggest that: (i) the physiologlcal expression of p2lWmm1 may be associated with cellular senescence of colorectal mucosa; (11) reduced expression of p21-m may participate in the progresslon of colorectal carcinoma; and (lii) p53-Independent paulway may be conelderably Involved In the inductlon of ~ 2 1 ~ ~ ~ ~ ~ ~ ~ ‘ .

Key wards: cdorectal adencarcinoma, colorectal adenoma, immunohistochemistty, p21 wMIIcIpl, p53, western blotting

Accumulations of various genetic alterations including oncogenes, tumor suppressor genes and DNA repair genes

Correspondence: Eiichi Tahara, MD, First Department of Pathology, Hiroshima Univers-ity School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734, Japan. Ernail: cetahara@mcai.med.hiroshima- u.ac.jp>

Received 19 November 1996. Accepted for publication 18 February 1997.

are involved in human carcin0genesis.l Recent studies have focused on the abnormalities in cell cycle regulators, which allow unbridled cell growth and div is i~n.~,~ Multiple cyclins and cyclin-dependent kinases (CDK) are positive regulators that progress the cell cycle; while several distinct protein inhibitors for CDK, such as p21wAF1/c1p1, ~ 2 7 ~ ~ ~ ‘ ~ ~ 1 6 ~ ~ and ~ 1 5 ~ = , potentially act as tumor suppressors.*-“ Rearrangement, deletion or mutation of the p16 and p15 genes have been reported in a majonty of human tumor cell

gene binds to the complex of cyclin E-CDK2 and cyclin DCDK4, inhibits the activity of, and prevents cells from entering the S pha~e.~o~. l~ p21 has also been shown to inhibit DNA replication through interaction with proliferating cell nuclear antigen (PCNA).14 p27 wAF7/cIp1 is a p53-inducible gene, whose promoter has a p53 binding site and the transcription is activated by wild-type but not mutated ~53.48~ p27wAF1c‘pf gene is identical to sdil, which was isolated from senescent fibroblasts and blocks DNA synthesis in young cells.I5 Although CDK inhibitors are candidate tumor suppressors, mutation of p27wAF1c’p1 gene is exceptional in human malignancies, while polymorphism of the gene at codon 31 in a highly conserved region is frequent in human tumors as well as their normal counterpa~ts.~~.~~

Loss of heterozygosity and mutation of p53 gene and abnormal accumulation of p53 are observed in 5040% of colorectal carcinomas.i8 We have recently demonstrated that the expression of p21 wAFIIclpl is correlated with the p53 gene status in gastric and colorectal carcinoma cell lines with some exception^.'^ On the other hand, recent reports have indicated that p21 wAF1/clpl is expressed in a variety of human tumor tissues but is not necessarily correlated with p53 gene mutations.- In in vifro experiments, the expression of p21wAF1K1p1 has been shown to be induced not only by the p53-dependent manner but also by p534ndependent p a t h ~ a y s . ~ ~ ~ ~ ~ The growth inhibition of transforming growth factor (TGF)-pi is associated with p53-independent induction of p21 ?5 El-Deity ef a/. examined the expression of p21 and

Jines.8.10.12

Aproduct of

p21 expression in colorectal tumors 471

p53 in a small number of colorectal adenomas and adenocarcinomas and found loss of topological control of p21 expression with a partial correlation with p53 gene abnormalities.20 However, no study of a large scale has been conducted to examine the expression of p21wAF1/c'p1 to elucidate the role of p21 in the development and progression of the colorectal carcinomas.

In the present study, we examined the expression of p21wAFi/c1p1 mainly by immunohistochemistry in 377 adenomas and 289 adenocarcinomas of the colorectum. We also analyzed the correlation of p21 expression with clinicopathological parameters as well as the expression of p53 protein.

MATERIALS AND METHODS

Tlssue specimens

A total of 289 adenocarcinomas and 377 adenomas of the colorectum were studied. They were obtained by surgery, endoscopical removal and biopsy at Hiroshima City Medical Association Clinical Laboratory (Hiroshima, Japan). They were fixed in 10% buffered-formalin and embedded in paraffin. One or two representative slides of the primary tumors, which included superficial, central and deep areas, were analyzed from each case. Among the adenocarcinoma cases, detailed clinicopathological findings could be obtained in 155 cases of surgery and endoscopical removal. The definitions of stage grouping, histological classification (grade of atypia), and depth of tumor invasion were made according to the criteria of the Japanese Research Society for Cancer of the Colon and Rectum.% (i) Depth of tumor invasion: m, mucosa including muscularis mucosae; sm, submucosa; and mp, muscularis propria. (ii) Stage grouping: 0, lesion limited to m and no metastasis; 1, lesion confined to sm and/or mp and no metastasis; 2, invasion deeper than mp but limited to the bowel wall and no metastasis; 3, extension to extracolorectal tissue and/or regional lymph node metastasis; and 4, metastasis to distant lymph node or organ and/or peritoneal dissemination regardless of depth of invasion.

For western blotting, tumor tissue samples were obtained at the time of surgery, immediately frozen in liquid nitrogen and stored at -8O'C. They included tumor tissues from superficial areas to deeply invasive parts, macroscopically. We confirmed histologically that tumor samples consisted mainly of carcinoma tissue.

p53 polyclonal antibody (CM-1) and anti-Ki-67 monoclonal antibody (MIB-1) were obtained from Novocastra (Newcastle upon Tyne, UK) and Medical and Biological Laboratories (Nagoya, Japan), respectively.

A modification of the immunoglobulin enzyme bridge technique (avidin-biotin peroxidase complex (ABC) method) was employed as described previ0usly.2~ Deparaffinized tissue sections were immersed in methanol containing 0.03% H20P for 30 min to block the endogenous peroxide activity. For detection of p21 and Ki-67, microwave pretreatment in citrate buffer was performed for 10x3 min to retrieve the antigenicity. The sections were then incubated with normal horse serum (diluted 1 :20) for 30 min to block the non- specific antibody binding sites. The sections were treated consecutively at room temperature with anti-p21 antibody (diluted 1 : 200), anti-p53 antibody (diluted 1 : 1OOO) or anti- Ki-67 antibody (diluted 1 : 100) for 90 min, biotinylated anti- mouse or rabbit IgG horse serum (diluted 1 : 100; Vector Laboratories, Burlingame, CA, USA) for 30 min, and avidin DH-biotinylated horseradish peroxidase complex (vectastain ABC kit; Vector) for 30 min. Peroxidase staining was performed for 10-15min using a solution of 3,3'- diaminobenzidine tetrahydrochloride in 50 mmoVL Tris-HC1 (pH 7.5) containing 0.001% H,O,. The sections were weakly counterstained with 0.1 % hematoxylin.

All of the immunostained slides were observed by three pathologists independently to make the grading as objective as possible. After agreement on the grades, we analyzed them using other parameters. Only the nuclear staining was regarded as positive for all three molecules (p21, p53, Ki-67).

For adenomas and superficial carcinomas, entire stained sections were observed. For large tumors of advanced cases, at least ten fields including superficial, central and deeply invasive areas were observed, and the number of stained cells and staining intensity were estimated. The immunoreactivity was graded as (-) to i-t+ as follows: for p21, (-), almost no positive cells; (+), 525% of tumor cells showed weak to moderate immunoreactivity; ++, 2550% of tumor cells showed moderate immunoreactivity or 10-50% of the tumor cells showed intense immunoreactivity; +ft, over 50% of tumor cells showed intense immunoreactivity. For p53, (-), almost no positive cells; (+), 525% of tumor cells showed immunoreactivity; ++, 2540% of tumor cells showed immunoreactivity; +++, over 50% of tumor cells showed immunoreactivity. Grades t+ and -I++ were regarded as strongly positive.

Western blotting lmmunohistochemistry

A monoclonal antibody to p21w*1Etp1 (sc-187) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA. USA). Anti-

The extracts of tissue samples were prepared as has been described elsewhere.n These extracts have mainly nudear and cytoplasmic fractions. Protein concentration was

472 W. Vasui et a/.

determined by Bradford protein assay (Bio-Rad. Richmond, CA. USA) using bovine serum albumin as a standard

Westem blotting was carried out as described elsewhere 27 One hundred micrograms of protein samples were applied to 1290-polyacrylamide gel electrophoresis followed by electrotransference onto a nitrocellulose filter Anti- P21 c'pl antibody (sc-187) and anti-p53 antibody (CM-1) were used By using the ECL western blotting detection system (Amersham, Aylesbury. UK), the immuno-complex was visualized

cells in the proliferative zone did not show p21 expression p53 immunoreactivity was not detected in normal colorectal mucosa (data not shown)

p21-positive cells were detected in 206 (55%) of the 377 adenomas. of which 18% were strongly positive (grade of immunoreactivity, ++ and +++, Table 1) The incidence of p21WAF1 c l p l expression did not correlate with the degree of atypia in adenomas There was a tendency for p21 to be preferentially expressed in adenoma cells located in the surface area This tendency was most prominent in adenomas with mild to moderate atypia

RESULTS p21 wAF1cipl expression in colorectal adenocarcinomas

~ 2 1 ~ ~ ' " ' ~ ' expression in non-neoplastic mucosa and adenomas of colorectum

The expression of p21 clp' was observed in the surface epithelial cells of normal colonic mucosa (Fig 1) These cells expressing p21 were negative to Ki-67 antigen, which is a marker of proliferating cells (cells at G1 and S phase) 29 The

p21 -positive tumor cells were observed in 73 (62%) of the 11 8 adenocarcinomas in adenoma and 117 (6S0/0) of the 171 adenocarcinomas without an adenoma component, respectively (Table 1). The incidence of strongly positive cases was significantly higher in the adenocarcinomas in adenoma (28%) as well as in the adenocarcinomas without an adenoma component (26%); than in the adenomas (1 8%);

Figure 1 they are negative to Ki-67 (c) lrnrnunostaining of p2lWAF' c'pl in adenorna of the colon The adenorna cells in the surface area are positlve to p21

lrnrnunostaining of (a) p21wAF1~c'p' and (b) Ki-67 in normal colonic rnucosa The surface epithelial cells are positive to p21 whereas

p21 expression in colorectal tumors 473

(P4.05). The immunoreactivities to p21 were localized in the nuclei of the tumor cells (Figs 1,2). Since most of the adenocarcinomas were of the well differentiated or

Table 1 adenocarcinomas

Expression of p21 in colorectal adenomas and

Histological types

Adenoma Mild+ Moderate Severe

Adenocarcinoma in adenoma

Adenocarcrnoma

~ ~

p21 positive lesions No cases Positive' Strongly positive'

377 206 (55%) 68 (1 8%) 46 22 (48%) 6 (1 3%)

112 61 (54%) 24 (21%) 219 123 (56%) 38 (1 7%)

118 73 (62%) 33 (28%)' 117 (68%): 44 (26%)' 171

'Imrnunoreactivity was graded (-), (+), ++ and +++ as descnbed in matenals and methods Positive, grades (+), ++ and +++, Strongly positive. grades ++ and +++ 'Grade of atypia was classified into three groups according to the critena of the Japanese Research Society for Cancer of the Colon and Rectum The incidence was significantly higher than that of adenomas (fa) 01 by Chi-squared test) 'The incidence was significantly higher than that of adenornas (Pa) 05 by Chi-squared test)

moderately differentiated type, no difference in the incidence of p21 expression was found by histological type.

We analyzed the relationship between p21 expression and clinicopathological parameters of 155 carcinoma cases obtained by surgery and endoscopic removal, in which full information could be obtained. As shown in Table 2, the incidence of strongly positive cases was significantly higher in stages 0 (lesion limited to the mucosal layer and no metastasis), 1 (lesion confined to submucosa andor muscularis propria and no metastasis), and 2 (invasion deeper than muscularis propria but limited to the bowel wall and no metastasis) carcinomas, than in stages 3 and 4 carcinomas (metastases in lymph nodes or distant organs regardless of depth of invasion; P4.05). As to the depth of tumor invasion, a similar correlation was detected. The expression of p21 was most prominent in the carcinomas that involved mucosa, submucosa and muscularis propria, but not in those carcinomas that went deeper than the muscularis propria. Although heterogeneity in p21 staining within a tumor was noticed in some cases, no obvious tendency was

Figure 2 lmmunostaining of (left panel) ~ 2 1 ~ " ~ ' ~ ' ~ ' and (right panel) p53 in colorectal adenocarcinomas (a,b) An example of p21 -positive/p53- positive carcinoma (c,d) An example of p21 -negative/p53-positive carcinoma

474 W. Yasui et al

detected between the location of tumor cells and p21 immunoreactivity Twenty-six percent of the carcinomas without lymph node metastasis expressed p21 strongly, whereas no carcinomas with metastasis were strongly positive to p21, the incidence being significantly different ( P 4 05).

Relationship between expression of p21 and p53

First. the expression of p21WAF1CIP' and p53 protein was examined in colorectal carcinomas by western blotting As shown in Fig 3. three of the 10 carcinomas expressed p21, while SIX carcinomas expressed p53 Among them, two cases (cases 42 and 195) showed expression of both p21 and p53 simultaneously No obvious correlation between p21 and p53 was detected The expressions of p21 and p53 as detected by western blotting was compatible with the irnmuno- histochemistry results of most cases examined (Table 3), although some exceptions existed possibly due to hetero- geneity of tumor cells and the difference in sensitivity between the two methods

We next examined the correlation between expressions for p21 and p53 immunohistochemically Of the 242 carcinomas

Table 2 Correlation between p21 expression and clinicopathological findings of colorectal carcinoma

in which p21 and p53 could be examined, 43% showed an inverse correlation between p21 and p53 (Table 4); that is, 31 Yo were p21 -positive and p53-negative. while 12% were p21 -negative and p53-positive (Fig. 2c.d). Thirty-three percent of cases showed expressions for both p21 and p53 (Fig. 2a,b). Some carcinomas consisted of both a p53- positive clone and a p53-negative clone; and p53-positive tumor cells were sometimes negative to p21. and vice versa, even within the same tumor. We then analyzed the relationship between p21 and p53 in biopsy specimens, in which a small area of the tumors is observed and heterogeneity of p53 can be partly excluded. However, as shown in Table 4, 48% (34+14%) of which biopsy cases showed an inverse correlation between the two, which was nearly the same as the incidence for all cases. There seemed to be no significant correlation between the expression of p21 wAF1'cIpl and the abnormal accumulation of p53 protein.

No cases

Stage' 0 111 1 18' 2 10 3 7 4 9

m 111

Beyond mp 26

Negative 141

Depth of invasion+

sm. mp 18'

Lymph node metastasis

p21 positive lesions Positive' Strongly positive'

-

66 (59'10) 30 (27?'0)* 13 (72%) 4 (22%)' 7 (70%) 2 (20%)' 7 (57%) 0 (OYO) 6 (67%) 0 (0%)

66 (59'/0) 30 (27%)" 13 (72%) 4 (22%)" 17 (65%) 2 (8%)

87 (62%) 36 (26'h)n Positive 14 9 (64%) 0 (0%)

'Immunoreactivity was graded as (-), (+), ++ and +++ as described in materials and methods. Positive. grades (+), ++ and +++; Strongly positive, grades ++ and +++.

'Graded according to the criteria of the Japanese Research Society for Cancer of the Colon and Rectum. m. mucosa including muscularis mucosae; sm. submucosa; mp. muscularis propria. Xonsisting of 18 carcinomas limited to sm and two carcinomas invading into mp. SThe incidence was significantly higher in carcinomas of stages 0. 1 and 2 than in those in stages 3 and 4 (P4.05 by Fisher's exact test). "The incidence was significantly higher in carcinomas invading beyond muscularis propria than in those confined within muscularis propria or more superficial (P4.05 by Fisher's exact test). "The incidence was significantly higher in carcinomas without lymph node metastasis than in those with metastasis (P4.05 by Fisher's exact test).

Figure 3 Western blot analysis of p21WAF"CiP' and p53 in colorectal adenocarcinomas. The protein lysates were prepared from freshly frozen tissues and western blotting was performed as described in Materials and Methods. The numbers above the panel indicate the case number.

Table 3 western blotting and immunohistochemistry

Case no Western' IHC'

Detection of p21 and p53 in colorectal carcinomas by

P2 1 P53 Western' IHC' _ _ ~

- 3 ++ + + 32 + ++ ++ 39 NE + NE 42 + ++ ++ +++ 51 90

- -

- - ~

- - ~

- - 109 + +++ 181 + ++ 186 ++ ++ 195 ++ ++ ++ ++

-

~ ~

'The levels of p53 shown in Fig 3 were graded as (-) (+) ++ and +++ according to the intensity 'IHC. immunohistochemistry lrnrnunoreactivity was graded (-) (+ I , ++ and +++ as described in materials and methods NE. not examined

p21 expression in colorectal tumors 475

Table 4 Expression of p21 and p53 in colorectal adenocarcinornas

p21 -positive' p53-positive

p21 -positive p21 -negative p21-negative p53-negative p53-positive p53-negative Total

All cases 79 (33%) 75 (31 Yo) 30(12%) 58 (24%) 242 Biopsy cases 35 (34%) 35 (34%) 15 (14%) 19(18%) 104

'Irnrnunoreactivity was graded as (-), (+), ++and +i+ as described in materials and methods. Positive includes grades of (+), ++and +I+: and negative includes grade of (-).

DISCUSSION

In normal colorectal mucosa, p21-positive epithelial cells were detected in the surface area of the crypts, while no cells positive to Ki-67 were detected in this area. The former are cells that are fully differentiated and are going to strip off the lumen by a renewal mechanism. p21wAF1mc'p1 expression may be associated with the cellular senescence of colorectal mucosa. It has been reported that senescent fibroblasts express p21 at higher levels than young Since normal epithelial cells were negative to p53 alterations, the expression of p21 must be induced in a p53-independent manner. This is compatible with the findings reported by El-Deity et a/. that the expression of p21 in normal gastrointestinal mucosa is topologically controlled by a p53- independent mechanism.20 The results of the present study are also consistent with the fact that the expression of mouse p21 correlates with the terminal differentiation of multiple cell lineages, such as skeletal muscle, skin and nasal epithelium, in a p53-independent

In adenomas, 55% contained p21 -positive cells. There was a tendency for p21 to be preferentially expressed in the adenoma cells located in the surface area. This tendency was most prominent in adenomas with mild to moderate atypia. However, unlike normal mucosa, the surface area of the adenomas contained a varying number of proliferating cells positive to Ki-67. This disordered pattern of p21 expression may be associated with the early stages of tumorigenesis of the colorectum. The pattern of p21 expression was not different between tubular adenomas and those that were tubulovillous. Since adenoma samples examined here included a small number of pure villous adenomas, we could not detect the special characteristics of p21 expression in this type of adenoma.

More than 60% of colorectal carclnomas expressed p21 wAF1mlpl and 27% of these were strongly positive to p21. Since p21wAF''Cc'p1 is a negative regulator for cell cycle progression,45 we expected an inverse correlation to exist between p21 expression and highgrade malignancy of colorectal cancer. As expected, a decreased expression of p21 was detected in cases at an advanced stage, deeply invasive carcinomas, and cases with lymph node metastasis. No cases with a strong expression to p21 were found in stage 3 and 4 carcinomas as well as carcinomas with metastasis.

The incidence of strong expression to p21 was significantly lower in deeply invasive carcinomas (beyond muscularis propria) than in superficial carcinomas. These findings suggest that the decreased expression to p21 may participate in the invasion and metastasis of colorectal carcinomas through the deregulation of cell cycle progression. We have already reported that the number of proliferating cells monitored by Ki-67 antibody is greater in the deeply invasive carcinomas than in the superficial carcinomas.36 We need to confirm the participation of reduced p21 in metastasis by comparing the expression of p21 between primary and metastatic tumors.

On the other hand, we have recently found that the expression of p21 correlates with the malignant behavior of gastric carcinomas directly, rather than inversely.= Similar trends have been reported in carcinomas of the ovary?' The proliferation and progression of cancer cells are regulated not only by one cell cycle regulator, p21wAFiK1p1, but also by a balance of various negative and positive regulators. It seems likely that the role of cell cycle regulators in the development and progression of cancer may have organ specificity. One example is the case of cyclin E; the expression of cyclin E is implicated by the early progression of colorectal carcinomas, while abnormalities of cyclin E in gastric carcinomas confer malignant behavior, such as deep invasion and metastasis.-

Although p27w'Mpt was first identified as a p53-inducible gene, the expression of is also known to be induced by a p53-independent pathway. We have recently reported that only half of the gastric carcinoma tissues show an inverse correlation between p21 and p53, while the level of p21 expression is closely related with the p53 gene status in gastric carcinoma cell line.^.^^.^ In the present study, 45-48% of the colorectal carcinomas showed an inverse correlation between the expression of p21 and the abnormal accumulation of p53, suggesting no direct correlation between the two. Although the present study is an imrnunohistochernical one, we have confinned a positive correlation between p53 immunoreactivity (from our staining conditions without microwave pretreatment) and p53 gene abnormalities in more than 70% of cobrectal and gastric carcinomas.1a*36 These findings, overall, suggest that p53- independent induction of p21wAF1cc"1 expression may be considerably involved in colorectal carcinomas.

476 W. Yasui eta/.

Alternatively, the expression of p21 can be regarded to be regulated by a p53dependent pathway in about a half of colorectal carcinomas. We have previously found that the incidence of loss-of-heterozygosity of the p53 gene and the abnormal accumulation of p53 protein increases as the tumor stage advances in colorectal carcinomas, whereas no such tendency has been detected in gastric carcinomas.1R35.36This difference may partly explain the aforementioned inverse correlation between colorectal and gastric carcinomas and the role of p21 in tumor progression.

p53-independent induction occurs due to various agents related to differentiation, growth arrest and apoptosis, including TGF-p."25.37 p21 expression is also induced by growth factors, such as platelet-derived growth factor, fibroblast growth factor and epidermal growth factor, independently of the p53 gene.= In the present study, reduced expression of p21 was detected in advanced colorectal carcinomas. We need to look at the expression of these factors with regard to the control mechanism of p21 induction in colorectal carcinomas. Since mutations of the p27 gene are exceptional, it is unlikely that the genetic abnormalities of p27 account for the reduction in its ex~ ress ion .~~ .~~ It has been recently reported that hypenethylation of the 5'CpG island is responsible for silencing of the p76 gene in human cancers.38 It would be of interest to examine the methylation status of the p27 gene in advanced colorectal carcinomas.

ACKNOWLEDGMENTS

We are grateful to staff members at the Pathology Division, Hiroshima City Medical Association Clinical Laboratory (Hiroshima, Japan) for their skillful technical assistance. This study was supported in part by a Grants-in-Aid for Cancer Research from the Ministry of Education, Science and Culture of Japan, and from the Ministry of Health and Welfare of Japan.

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