Cyclin D1 and p21 in ulcerative colitis-related inflammation and epithelial neoplasia: a study of aberrant expression and underlying mechanisms

It is unclear whether and how cyclin D1 and/or p21(WAF1/CIP1) dysregulation contribute to ulcerative colitis (UC)-related inflammation and colorectal carcinogenesis. Cases of quiescent UC (QUC; n = 15), active UC (AUC; n = 23), UC-related dysplasia (n = 35) and UC-related colorectal adenocarcinomas (CRCs; n = 11) were studied with cyclin D1 and p21(WAF1/CIP1) immunohistochemistry. The CRCs were also studied with beta-catenin, bcl2, and p53 immunohistochemistry, p53 and k-ras mutation analyses, and cyclin D1 gene fluorescence in situ hybridization. QUC showed cyclin D1 (negative/weak staining) and p21(WAF1/CIP1) (surface epithelial and upper-third crypt staining) expression similar to that of normal colorectum. Moderate or strong cyclin D1 immunostaining was seen in 9% of AUC cases, 40% of dysplasia cases, and 36% of UC-related CRCs. Although these carcinomas showed neither cyclin D1 gene amplification nor any association between k-ras mutation and cyclin D1 overexpression, the latter was closely related to nuclear beta-catenin expression. Increased lower-third crypt p21(WAF1/CIP1) staining was seen in 57% of AUC cases; decreased upper-third crypt p21(WAF1/CIP1) staining, in 23% of dysplasia cases; and absent or weak p21(WAF1/CIP1) staining, in 55% of UC-related CRCs. The latter change was always associated with p53 mutation but could not be related to p53 or bcl2 expression. In conclusion, AUC shows up-regulated cyclin D1 and p21(WAF1/CIP1) expression. Cyclin D1 up-regulation and p21(WAF1/CIP1) down-regulation occur early in UC-related carcinogenesis. Cyclin D1 up-regulation is less common in UC-related CRCs than in sporadic CRCs, and is related to beta-catenin nuclear signaling. p21(WAF1/CIP1) down-regulation is seen at an equal or higher frequency among UC-related CRCs compared with sporadic CRCs and is attributable to p53 mutation.     

Increased cyclin D1 expression in cancer of the ampulla of Vater: relevance to nuclear beta catenin accumulation and k-ras gene mutation.

AIMS: Several studies have reported that dysregulation of beta catenin or k-ras mutation promotes cyclin D1 expression. This study investigated the relation between cyclin D1 expression and clinicopathological parameters in carcinoma of the ampulla of Vater (CAV), and also assessed the relation between increased cyclin D1 expression and beta catenin/k-ras status in this series. METHODS: Thirty CAVs were evaluated for cyclin D1 expression by immunohistochemistry in relation to patient clinicopathological features. Aberrant beta catenin expression and k-ras mutation were also investigated by immunostaining and direct sequencing, and related to cyclin D1 expression. RESULTS: Increased cyclin D1 expression was seen in 17 of 30 CAVs and was significantly correlated with tumour cell proliferation and disease free survival time (p = 0.018, p = 0.018, respectively). Nuclear accumulation of beta catenin was found in nine of 30 cases, including four cases with missense mutations in exon 3 of CTNNB-1, and was significantly correlated with increased cyclin D1 expression (p = 0.003). k-ras gene mutation was detected in 12 of 30 cases, and was also significantly correlated with increased cyclin D1 expression (p = 0.026). Overall, 14 of 17 CAVs with increased cyclin D1 expression showed nuclear accumulation of beta catenin and/or k-ras mutation. CONCLUSIONS: Increased cyclin D1 expression appears to be associated with tumour proliferation and poorer clinical outcome in CAV. It is also associated with both aberrant beta catenin expression and k-ras mutation. These results are consistent with the in vitro data that cyclin D1 can be transactivated by activated beta catenin-T cell factor/LEF and k-ras pathways.     

Nuclear beta-catenin expression is rare and its potential association with short survival in colorectal signet-ring cell carcinoma.

Colorectal signet-ring cell carcinoma (SRCC) is a rare cancer and the prognosis is usually very poor. The biologic pathways involved in its oncogenesis are unknown. beta-catenin, a key target in the Wnt-signaling pathway, is recognized to play an important role in the carcinogenesis in conventional colorectal carcinoma. This study explores the involvement of Wnt-signaling molecules beta-catenin and cyclin D1, cell cycle regulators cyclin D3, proliferative index Ki-67, apoptotic index, and angiogenic indicator CD31 in 20 colorectal SRCC paraffin-embedded specimens. Results showed that there were 2 specimens with nuclear beta-catenin and higher expression of cyclin D1 than the remaining 18 specimens. Surprisingly, those 2 patients had a much shorter survival of 6 months than the remaining 15 patients, who had around 24 months. Moreover, all colorectal SRCC specimens had an overexpression of cyclin D1, cyclin D3, and Ki-67, as well as much more angiogenesis and apoptosis than adjacent normal epithelial tissues. The authors make the preliminary comment that nuclear beta-catenin is a rare phenomenon in colorectal SRCC, but the involvement of it may indicate a worse prognosis with shorter survival than colorectal SRCC without nuclear beta-catenin expression. Besides, overexpression of cyclin D1, cyclin D3, Ki-67, and increased angiogenesis and apoptosis may play a vital role in promoting colorectal SRCC development.     

Expression of cyclin Ds in relation to p53 status in human breast carcinomas

 Cyclin D1 has been reported to be overexpressed in many tumours, including breast carcinomas. Cyclin D1 was first identified as a protooncogene (BCL1/PRAD1), and its overexpression was related to tumour proliferation. The product has also recently been identified as important in mediating cell cycle growth arrest via the p53 pathway in murin fibroblast cell lines. Ninety breast carcinomas previously analysed for p53 status were analysed for amplification of cyclin D1, D2 and D3 genes by Southern blot analysis and for protein expression by immunhistochemistry. In 10 samples gene amplification was detected at the cyclin D1 locus. No gene amplification was detected at the cyclin D2 and D3 loci. Immunoreactivity for cyclin D1 was detected in 38 (42.2%) tumour tissue samples. Fifty samples were immunostained for cyclin D2 and D3. Only 2 samples (4%) showed immunoreactivty for cyclin D2, and 9 samples (18%) for cyclin D3. Cyclin D1 protein overexpression was significantly more often found in tumours with wild type p53 and in tumours with higher grades of differentiation expressing ER. No association was seen between gene amplification of the cyclin D1 gene and p53 status. We conclude there is a relationship between wild type p53 and cyclin D1 protein overexpression in clinical material, indicating that cyclin D1 may be another downstream effector of p53. Received: 25 November 1997 / Accepted: 30 March 1998     

Cyclin D1 overexpression in N-methyl-N′-nitro-N-nitrosoguanidine-induced rat gastric adenocarcinomas

Changes in cell cycle regulation are involved in many human cancers, including gastric cancer. In the present study, cyclin D1 expression and localization were immunohistochemically analyzed in 23 N-methyl-N'-nitro-N-nitrosoguanidine-induced rat gastric adenocarcinomas and compared with findings for beta-catenin. Cyclin D1 nuclear overexpression was more frequently observed in tumors displaying nuclear (4/4=100%) and cytoplasmic (3/4=75%) beta-catenin accumulation than those with membranous (3/15=20%) localization (nuclear vs. membranous, P<0.02). In the former cases it was considered that cyclin D1 was induced with beta-catenin activation; in the latter, a direct or indirect pathway for cyclin D1 accumulation bypassing Wnt pathway might be involved. Cyclin D1 was also found to be accumulated in gastric glands within normal-looking mucosa, these perhaps representing preneoplastic lesions for cancers with membranous beta-catenin accumulation.     

Lack of cyclin D1 overexpression in gastric carcinogenesis

AIMS: Cyclin D1 overexpression was examined in early gastric carcinomas and precursor lesions with the following aims; (1) to assess the chronology of cyclin D1 overexpression in various stages of gastric carcinogenesis, (2) to correlate cyclin D1 overexpression with the Lauren type, the grade of differentiation and the type of growth pattern of the tumours and (3) to correlate cyclin D1 overexpression with clinical parameters, in particular lymph node metastasis and overall prognosis. METHODS AND RESULTS: Forty-five paraffin-embedded gastrectomy specimens from early carcinomas were examined for the presence of various precursor lesions. The Lauren type, the grade of differentiation and the type of growth pattern were reassessed for all early carcinomas. Cyclin D1 overexpression was examined using the monoclonal antibody DCS-6. Cyclin D1 overexpression was absent from all precursor lesions. Ten early carcinomas (22%) were cyclin D1 positive without significant differences when stratified according to Lauren type, grade of differentiation, type of growth pattern or lymph node status. Univariate analysis failed to show a significant difference in 5-year surival rate between cyclin D1 positive and negative early carcinomas (90% vs. 94%). CONCLUSIONS: Cyclin D1 protein overexpression does not play a role in the progression from normal to neoplastic gastric mucosa and does not discriminate between intestinal and diffuse type early gastric carcinomas of Caucasian origin. Moreover, mechanisms other than cyclin D1 protein overexpression underlie the reported difference in biological behaviour of early gastric carcinomas with different types of growth pattern. Finally, although it appears that cyclin D1 does not have prognostic significance, studies on larger numbers, including advanced carcinomas, are warranted.     

Cyclin E gene (CCNE) amplification and hCDC4 mutations in endometrial carcinoma

Cyclin E overexpression occurs in a subset of endometrial carcinomas (ECs), but the molecular mechanisms underlying this alteration remain to be established. The present study has analysed amplification of the cyclin E gene (CCNE) and mutation in hCDC4, the gene coding for the F-box protein, which tags phosphorylated cyclin E for proteosomal degradation, to ascertain whether these alterations might be responsible for cyclin E overexpression in ECs. Cyclin E and p53 expression was studied by immunohistochemistry in eight atypical endometrial hyperplasias (AEHs), 51 endometrioid endometrial carcinomas (EECs), and 22 non-endometrioid endometrial carcinomas (NEECs). CCNE amplification was analysed by fluorescence in situ hybridization (FISH). Mutations in exons 2-11 of the hCDC4 gene were screened by PCR-SSCP-sequencing. Finally, the polymorphic marker D4S1610 was used to assess loss of heterozygosity (LOH) in the hCDC4 gene. Cyclin E overexpression was found in 26/81 (32%) cases and was associated with the histological type of the lesion, since it was not found in any AEHs but was present in 27% of EECs and 54.5% of NEECs (p=0.035). Cyclin E overexpression was associated with histological grade (p=0.011) and p53 immunostaining in EECs (p=0.033). CCNE amplification was found in 6 of 37 (16%) ECs examined. There was a significant association between CCNE amplification and the histological type of the lesion, since five (83%) of the six cases with amplification were NEECs (p=0.008). One EEC harboured an hCDC4 mutation: a CGA to CAA (Arg/Gln) change at codon 479. In addition, D4S1610 LOH was found in 7 of 23 (30%) informative cases analysed, but no correlation with cyclin E overexpression was found. However, the tumour with hCDC4 mutation also showed LOH. This is the first study demonstrating that cyclin E overexpression is associated with gene amplification in ECs, these alterations being more frequent in NEECs. Although hCDC4 exhibits a low mutation frequency in ECs overexpressing cyclin E, it seems to function as a tumour suppressor gene that is involved in endometrial carcinogenesis.     

Beta-catenin accumulation in the progression of human hepatocarcinogenesis correlates with loss of E-cadherin and accumulation of p53, but not with expression of conventional WNT-1 target genes

Beta-catenin integrates intracellular WNT signalling and the intercellular E-cadherin-catenin adhesion system. To date, little is known about the role of beta-catenin activation and nuclear accumulation in hepatocarcinogenesis. This study has analysed beta-catenin expression patterns in human dysplastic nodules (DNs), as well as in hepatocellular carcinomas (HCCs) in comparison with proliferation, expression of WNT-1 target genes, E-cadherin, and p53. One hundred and seventy HCCs and 25 DNs were categorized according to established criteria and analysed for the expression pattern of beta-catenin. Analysis of the proliferative activity and expression of E-cadherin, cyclin D1, MMP-7, c-myc, and p53 was performed on a representative subgroup of cases. All DNs lacked nuclear beta-catenin, while 36% of all HCCs were positive, with the number of nuclear stained cells ranging from less than 1% to more than 90%. Increasing nuclear accumulation of beta-catenin correlated with reduced membranous E-cadherin expression and nuclear p53 but not with proliferation. Cyclin D1, MMP-7, and c-myc expression was detected in 54%, 26%, and 65% of HCCs, respectively, but did not correlate with nuclear beta-catenin, proliferation, or grading. Sequence analysis of the beta-catenin gene revealed no detectable mutations in DNs, but mutations in the GSK-3beta binding site were present in 14.3% of the HCCs. In conclusion, this study has demonstrated that nuclear accumulation of beta-catenin is a frequent progression event in human hepatocarcinogenesis which correlates with nuclear p53 accumulation and loss of membranous E-cadherin, but not with the expression pattern of established WNT-1 target genes. It is hypothesized that the role of beta-catenin in human HCC differs significantly from its established function in colon carcinogenesis.     

beta-catenin nuclear expression correlates with cyclin D1 overexpression in sporadic desmoid tumours

The immunohistochemical expression of beta-catenin, cyclin D1, Ki-67 and PCNA was Examined in 38 cases of sporadic extra-abdominal or abdominal-wall desmoid tumours without familial adenomatous polyposis (FAP), to evaluate the hypothesis that the accumulated beta-catenin within the nuclei could affect the regulation of the cyclin D1 gene. There was a statistically significant correlation between beta-catenin accumulation and cyclin D1 overexpression (p=0.029). Each group with beta-catenin accumulation or cyclin D1 overexpression showed a higher PCNA-LI than those without, the difference being statistically significant (p=0.007, p=0.004, respectively). Differential PCR was also performed to detect amplification of the cyclin D1 gene and mutational analysis was undertaken for exon 3 of the beta-catenin gene. Amplification of the cyclin D1 gene was observed in 13 out of 22 cases (59.1%). There were nine-point mutations in 7 out of 18 cases (38.9%). The distribution of beta-catenin mutation fell within a wide range, from codon 21 to codon 67. In conclusion, beta-catenin nuclear expression correlated with cyclin D1 overexpression in sporadic desmoid tumours, which could be an in vivo model system for the APC-beta-catenin-Tcf pathway. In addition, beta-catenin mutations in desmoid tumours occurred at an unusually wide range of sites within the gene.     

Immunohistochemical expression of cyclin D1, E2F-1, and Ki-67 in benign and malignant thyroid lesions

Cyclin D1 and E2F-1 proteins are essential for the regulation of the G1/S transition through the cell cycle. Cyclin D1, a product of the bcl-1 gene, phosphorylates the retinoblastoma protein, releasing E2F-1, which in turn activates genes involved in DNA synthesis. Expression patterns of E2F-1 protein in thyroid proliferations have not been reported. This study used monoclonal antibodies for cyclin D1 and E2F-1 proteins to immunostain sections of normal thyroid, hyperplastic (cellular) nodules, follicular adenomas, follicular carcinomas, and papillary carcinomas. The proliferation rate was examined using an antibody specific for the Ki-67 antigen. Fluorescence in situ hybridization (FISH) methods and chromosome 11-specific probes were also employed to determine chromosome copy number and to assess for evidence of amplification at the 11q13 locus in papillary and follicular carcinomas with cyclin D1 overexpression. Concurrent overexpression of Ki-67, cyclin D1, and E2F-1 was found in the majority of benign and malignant thyroid lesions, compared with normal thyroid tissue. Cyclin D1 up-regulation was not due to extra copies of chromosome 11, or bcl-1 gene amplification. Malignant tumours showed the highest expression for all three markers, particularly papillary carcinomas. E2F-1 was detected at the same or slightly lower levels than cyclin D1. It was only found when cyclin D1 was overexpressed. Because cyclin D1 normally activates E2F-1, up-regulation of cyclin D1 may lead to E2F-1 overexpression in benign and malignant thyroid lesions.     

Expression of Wnt-signaling pathway proteins in intraductal papillary mucinous neoplasms of the pancreas: a tissue microarray analysis

Abrogation of the Wnt-signaling pathway is implicated in the carcinogenesis of several malignancies, especially colorectal cancer where up to 90% of cases are thought to have impaired Wnt signaling. It is less frequently involved in conventional ductal pancreatic adenocarcinoma. This pathway has not been explored in intraductal papillary mucinous neoplasms (IPMNs) of the pancreas previously and formed the basis of this study. A tissue microarray of 18 cases of IPMN was stained for proteins involved in the Wnt pathway: adenomatous polyposis coli (APC), pan-beta-catenin, axin 2, glycogen synthase 3alphabeta and 3beta, c-myc, E-cadherin, and cyclin D1. The IPMNs were classified as 8 adenomas, 3 borderline, and 7 cases with carcinoma in situ and/or invasive carcinoma, occurring in 13 females, and the overall age range was 45 to 73 years. Immunohistochemical analysis showed nuclear beta-catenin staining in 7 (39%) of the 18 cases. The cases with nuclear beta-catenin localization included 1 adenoma, 2 borderline IPMN, and 4 carcinomas in situ and/or invasive carcinomas. Seven cases showed absence of APC immunostaining and these included 4 cases with nuclear beta-catenin localization. Fourteen cases displayed marked diffuse up-regulation of c-myc protein, and 12 cases also showed diffuse cyclin D1 protein overexpression. E-cadherin expression was intense and membrane in location (comparable to normal tissue) in 6 of 8 adenomas (no tissue was available in 1 case). Decreased E-cadherin staining was noted in 8 cases where tissue was available for assessment. There was progressive decrease in membrane staining of E-cadherin in 2 of 3 borderline lesions, 1 of 2 carcinomas in situ, and 4 of 5 invasive carcinomas. All other immunostains were either normal in distribution or did not show any correlation with beta-catenin or clinicopathologic parameters. In conclusion, 7 (39%) of 18 cases of IPMN in this study demonstrated abnormal localization of beta-catenin, 4 of which also lacked APC expression. Of 5 carcinomas arising in IPMN, 4 displayed a decrease in E-cadherin expression. There was also a trend for the higher grades of IPMN to show nuclear localization of beta-catenin. These findings suggest that a proportion of cases of IPMN may show abnormalities in the Wnt-signaling pathway with consequent altered expression of downstream related proteins.     

Cyclin D1 in invasive breast carcinoma: favourable prognostic significance in unselected patients and within subgroups with an aggressive phenotype

Aims: To study the clinicopathological and prognostic value of cyclin D1 overexpression in patients with breast carcinoma. Methods and results: Immunohistochemistry was performed on paraffin‐embedded tissue specimens from 290 invasive breast carcinomas to detect the proteins cyclin D1, oestrogen receptor (ER), progesterone receptor (PR), p53, c‐erbB2, and topoisomerase IIα (topoIIα). Cyclin D1 staining was quantified using a computerized image analysis method. Cyclin D1 overexpression characterized smaller, ER‐positive and PR‐positive tumours (P = 0.017, P < 0.0001, and P < 0.0001, respectively), of a lower histological and nuclear grade (P = 0.011 and P < 0.0001, respectively), and with reduced expression of topoIIα (P = 0.001) and p53 (P < 0.001). Cyclin D1 was found to have an independent favourable impact on the overall survival of both the unselected cohort of patients (P = 0.011) and of patients with ER‐negative and lymph node‐positive tumours (P = 0.034 and P = 0.015, respectively). In triple‐negative tumours, cyclin D1 overexpression was found to have independent favourable impacts on both overall and relapse‐free survival (P = 0.002 for both). Conclusions: This is the first immunohistochemical study to dissociate the advantageous prognostic effect of cyclin D1 overexpression from its association with ER expression, and to provide evidence that cyclin D1 overexpression may be a marker of prolonged survival in patient subgroups with aggressive phenotypes.     

Cyclin D1 is frequently overexpressed in microsatellite unstable colorectal cancer, independent of CpG island methylator phenotype

Aims: Cyclin D1 and cyclin‐dependent kinases are commonly activated in colorectal cancer. Microsatellite instability (MSI) and CpG island methylator phenotype (CIMP) are important molecular classifiers in colorectal cancer. The aim was to clarify the relationship between cyclin D1, MSI and CIMP. Methods and results: Among 865 colorectal cancers with MSI and CIMP data, 246 tumours (28.4%) showed cyclin D1 overexpression by immunohistochemistry. DNA methylation in p14 and eight CIMP‐specific promoters (CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1) was quantified by real‐time polymerase chain reaction (MethyLight). Both MSI‐high and CIMP‐high were associated with cyclin D1 overexpression (P < 0.0001). After tumours were stratified by MSI and CIMP status, the relationship between MSI‐high and cyclin D1 persisted (P ≤ 0.02), whereas the relationship between CIMP‐high and cyclin D1 did not. Cyclin D1 overexpression was correlated with BRAF mutation (P = 0.0001), p27 loss (P = 0.0007) and p16 loss (P = 0.02), and inversely with p53 expression (P = 0.0002) and p21 loss (P < 0.0001). After stratification by MSI status, the inverse relationship between cyclin D1 and p21 loss still persisted (P < 0.008). Conclusions: Cyclin D1 activation is associated with MSI and inversely with p21 loss in colorectal cancers. Cyclin D1 may play an important role in the development of MSI‐high tumours, independent of CIMP status.     

Immune recognition of cyclin B1 as a tumor antigen is a result of its overexpression in human tumors that is caused by non-functional p53

Cyclin B1, which plays a key role in the control of cell cycle progression from G(2) through M phase, was recently identified by us as a tumor antigen recognized by human T-cells. To understand what makes this normal molecule antigenic, we compared its expression in malignant versus normal cells. Immunohistology showed overexpression of cyclin B1 protein in tumors compared to surrounding normal tissue and localization in the cytoplasm rather than the nucleus. Cyclin B1 is overexpressed at protein and mRNA level in many tumor cell lines including breast, lung, colorectal carcinoma, lymphoma and leukemia. While overexpressed in tumor cells at all stages of the cell cycle, its expression still peaks at G(2)/M phase, as it does in normal cells. We compared cyclin B1 expression in two cell clones derived from the same colorectal tumor cell line, one wild type for p53 (HCT116p53(+/+)) and one with deleted p53 (HCT116p53(-/-)). HCT116p53(+/+) cells had undetectable (normal) level of cyclin B1 protein, while HCT116p53(-/-) cells showed overexpression. When reconstituted with p53, HCT116p53(-/-) cells reverted to normal cyclin B1 expression. We conclude that p53 plays an important role in cyclin B1 regulation and that tumors with mutated p53 will be good candidates for cyclin B1 based immunotherapy.     

Intratumoral heterogeneous expression of p53 correlates with p53 mutation,Ki-67, and cyclin A expression in endometrioid-type endometrial adenocarcinomas

To further elucidate the significance of p53 mutation in endometrial carcinoma, we investigated it in endometrioid-type endometrial carcinomas showing intratumoral heterogeneous p53 expression. In addition, we also examined the correlation of p53 mutation and cyclin A expression, because we previously reported a topological correlation between the expression of p53 and cyclin A. The p53 mutation in exons 5–8 in 54 cases of endometrial carcinoma showing immunohistochemical expression of p53 was examined using microdissected tissue DNAs. Of the 54 p53-positive endometrial carcinomas, 23 (43%) had p53 mutation with a tendency in histologically higher grade tumors. Ten of the 54 showed a heterogeneous p53 expression, and in 9 of the 10 cases, p53 mutation was present only in p53-positive sites, which were often found in histologically less differentiated areas with elevated Ki-67 in the same tumor. Cyclin A expression was topologically observed in p53-positive areas; however, it was noted in both tumors with (12/23, 52%) and without (18/31, 58%) p53 mutation. These results suggest that p53 mutation is a late event and plays an important role in the acquisition of malignant potentials in endometrioid-type endometrial adenocarcinomas. Unexpectedly, accumulation of the p53 protein itself may be important in cyclin A overexpression.