The role of cyclins and cyclins inhibitors in the multistep process of HPV-associated cervical carcinoma

BACKGROUND: Human papillomavirus (HPV) types 16 and 18 are associated with cervical carcinogenesis. This is possibly achieved through an interaction between HPV oncogenic proteins and some cell cycle regulatory genes. However, the exact pathogenetic mechanisms are not well defined yet. METHODS: We investigated 110 subjects (43 invasive squamous cell carcinoma (ISCC), 38 CIN III, 11 CIN II, 18 CIN I) confirmed to be positive for HPV16 and/or 18 as well as 20 normal cervical tissue (NCT) samples for abnormal expression of cyclin D1, cyclin E, CDK4, cyclin inhibitors (p21 (waf), p27, p16 (INK4A)) and Ki-67 using immunohistochemistry and differential PCR techniques. RESULTS: There was a significant increase in the expression of Ki-67, cyclin E, CDK4, p16 (INK4A) (p=0.003, 0.001, 0.001) and a significant decrease in p27 (Kip1) from NCT to ISCC (p=0.003). There was a significant correlation between altered expression of p27 (KIP1) and p16(INK4A) (p<0.001), cyclin D1 and CDK4 (p=0.001), cyclin E and p27 (Kip1) (p=0.011) in all studied groups. In ISCC, there was significant relationship between standard clinicopathological prognostic factors and high Ki-67 index , increased cyclin D1 and cyclin E, reduced p27 (Kip1) and p21 (waf). CONCLUSION: 1) Aberrations involving p27 (KIP1), cyclin E, CDK4 and p16 (INK4A) are considered early events in HPV 16 and 18-associated cervical carcinogenesis (CINI & II), whereas cyclin D1 aberrations are late events (CINIII & ISCC) 2) Immunohistochemical tests for p16 (INK4A) and cyclin E could help in early diagnosis of cervical carcinoma 3) Only FIGO stage, cyclin D1, p27 (Kip1) and Ki-67 are independent prognostic factors that might help in predicting outcome of cervical cancer patients.     

Frequent disruption of the RB1 pathway in diffuse large B cell lymphoma: prognostic significance of E2F-1 and p16INK4A.

In the present study, we analysed 34 de novo diffuse large B cell lymphoma (DLCL) from a population-based lymphoma registry for alterations of the RB1 pathway at the genetic (RB1 and CDK4) and protein (pRb, cyclin D1, cyclin D3, CDK4, and E2F-1) level. The results were correlated with the data from our previous studies of CDKN2A deletion and hypermethylation, other p53 pathway components, p27Kip1 expression, and proliferation, as well as with clinical outcome, including prognosis. We found aberrant pRb expression in four (12%) of 34 DLCLs. One of these had a point mutation in intron 3 10 bp downstream of exon 3 generating a novel splice signal. Seven tumours (21%) showed cyclin D3 overexpression, including all three thyroid lymphomas (P = 0.006). Cyclin D3 overexpression and p16INK4A/pRb aberrations were mutually exclusive, supporting an oncogenic role for cyclin D3 in DLCL. p16INK4A inactivation, cyclin D3 overexpression, or aberrant pRb expression was identified in 18 of 34 DLCLs (53%). Combining these results with our previous p53 pathway studies showed that 82% of the de novo DLCLs had alterations of these pathways, and that both pathways were altered in 13 cases (38%). Low E2F-1 expression was associated with treatment failure (P = 0.020), and multivariate analysis of overall survival identified both low E2F-1 expression (relative risk = 6.9; P = 0.0037) and p16INK4A inactivation (relative risk = 3.3; P = 0.0247) as independent prognostic markers. These data support a role of E2F-1 as tumour suppressor gene in lymphoma and strongly suggest that the RB1 and p53 pathways are important in the development of de novo DLCL. Furthermore, low E2F-1 expression and p16INK4A inactivation may serve as prognostic markers for patients with this type of lymphoma.     

Alterations of the cyclin D1/pRb/p16(INK4A) pathway in multiple myeloma.

The retinoblastoma protein (pRb), p16(INK4A), D-type cyclins, and their partners cyclin-dependent kinase (CDK) 4 and 6 constitute a G(1) regulatory pathway commonly targeted in tumorigenesis. Several malignancies show a reciprocal correlation between genetic alterations of single members of the pRb pathway. Therefore, we determined the frequency of Rb deletions and cyclin D1 alterations by fluorescence in situ hybridization as well as 5' CpG island hypermethylation of the p16(INK4A)gene using methylation-specific polymerase chain reaction in bone marrow mononuclear cells from 82 individuals with plasma cell disorders. Alterations in at least one of the components of the pathway were found in 75%. Cyclin D1 translocations or amplifications were detected in 14/82 (17.1%), Rb deletions at 13q14 in 23/82 (28%) of the cases, including three (3.6%) homozygous deletions. p16(INK4A) was hypermethylated in 33/57 (57.9%) of the samples. Further analysis revealed a highly significant correlation between cyclin D1 alterations and extramedullar or leukemic myeloma manifestations (P = 0.014; Fisher's test). Whereas Rb deletions seemed to occur alternatively to cyclin D1 alterations, no reciprocal correlation was found between p16(INK4A) hypermethylations and cyclin D1 or Rb locus aberrations. Cyclin D1 locus alterations and Rb deletions were associated with a significantly worse prognosis whereas p16(INK4A) hypermethylation had no impact on survival. We conclude that cyclin D1 and Rb aberrations seem to occur as alternative events in plasma cell malignancies and contribute to clinical course and prognosis. In contrast, although p16(INK4A) hypermethylation is frequent, inactivation of p16(INK4A) seems not to be involved in the pathogenesis of plasma cell disorders.     

Alterations in the retinoblastoma pathway of cell cycle control in parathyroid tumors

Mutations in proto-oncogenes and tumor suppressor genes have been associated with tumor development and/or progression in many neoplasms. It has been reported that parathyroid tumors have deletions affecting the retinoblastoma gene (RB), and overexpression of cyclin D1 (Cyc D1). The aim of the present study was to evaluate the alterations in the components of the pRB pathway in parathyroid adenomas and parathyroid aggressive tumors, including patterns of expression of pRB, Cyc D1, and p16/INK4A. Paired normal and tumor DNA from 6 parathyroid adenomas and 5 aggressive tumors were analyzed for loss of heterozygosity (LOH) at the RB locus. The expression of pRB, Cyc D1 and p16 was studied in 4 adenomas and 5 aggressive tumors. RB LOH was found in 1 of 6 adenomas, and in 1 of 2 informative aggressive tumors. Immunohistochemical analysis revealed undetectable pRB in 4 of 5 aggressive tumors and presence of pRB in all adenomas. Conversely, Cyc D1 expression was found in 3 of 4 aggressive tumors, but was undetectable in the adenomas. Expression of p16 was identified only in one aggressive tumor. Thus, alterations in the pRB pathway seem to prevail in the aggressive form of parathyroid neoplasms. Our results warrant further investigation of these cell cycle regulators in order to determine their potential role as tumor markers in parathyroid tumors.     

Expression of cell cycle control proteins in normal epithelium, premalignant and malignant lesions of oral cavity

In this study, we examined the expression of cyclins, cyclin dependent kinase (CDKs) and CDK inhibitors by immunohistochemical analysis in 20 normal mucosa, 42 epithelial dysplasia (ED), and 117 oral squamous cell carcinoma. Neither Cyclin D1 nor CDK2 were detectable in normal tissue and ED. Their presence, however, was detectable in squamous cell carcinoma (SCCs) (Cyclin D1, 35.9%; CDK2, 66.7%). Cyclin E was detectable in 57.1% of severe ED and 62.8% of SCCs. For the CDK inhibitors, these proteins were detectable in all normal mucosa and most of the mild and moderate ED. For severe ED, expression of these proteins was not observed in some cases (p12(DOC-1), 14.3%; p16(INK4A), 28.6%; p27(KIP1), 7.1%). For SCCs, the expression of p12(DOC-1) was lost in 71.8%, p16(INK4A) in 69.2% and p27(KIP1) in 35.9%. These results suggest that elevated expression of cyclin D1, cyclin E, CDK2 and loss of p12(DOC-1), p16(INK4A) and p27(KIP1) may contribute to the multistep nature of oral carcinogenesis.     

Alterations of Rb, p16(INK4A) and cyclin D1 in the tumorigenesis of oral squamous cell carcinomas

Immunohistochemical analysis of Rb, p16(INK4A) and cyclin D1 expression was performed on 78 oral squamous cell carcinoma (SCC), 46 leukoplakia, and 20 normal mucosa. Rb and p16(INK4A) expression were observed in all normal mucosa and most of leukoplakia. Lack of Rb and p16(INK4A) was observed in 56.4 and 67.9% of SCC, respectively. The overexpression of cyclin D1 was not observed in normal mucosa and was observed in 35.9% of SCC. A strong reciprocal relationship between Rb and p16(INK4A) expression was observed in oral SCC, and all these SCC cases have at least one of the alterations in the Rb pathway.     

Frequent overexpression of cyclin D2/cyclin-dependent kinase 4 in Wilms' tumor

The expression status of the three cyclin D genes (CCND1, CCND2 and CCND3), the two cyclin D-dependent kinase genes (CDK4 and CDK6) and the p16(INK4a) gene was studied in a series of 47 Wilms' tumors, 16 normal mature kidneys and two fetal kidneys. We showed predominant overexpression of CCND2 and CDK4 compared to CCND1/D3 and CDK6 respectively. We found a specific correlation between relapse and CDK4 overexpression, but not CDK6 overexpression. We did not identify any methylation of the p16(INK4a) promoter. This suggests that dysregulation of CCND2 and CDK4 plays a specific role in WT tumorigenesis.     

p21 is associated with cyclin D1, p16INK4a and pRb expression in resectable non-small cell lung cancer

p21 (p21WAF1/CIP1) is involved in cell cycle regulation, as an inhibitor of cyclin dependent kinases (CDK2, CDK4 and CDK6). However, subsequent in vitro studies have suggested that p21 may influence this process by an additional mechanism, in particular through the regulation of cyclin D1 subcellular localisation. This study of primary resectable non-small cell lung cancer (NSCLC) was designed to examine p21 functions in association with the expression of cyclin D1 (including its subcellular localisation), p16INK4a and pRb. p21 expression was examined in 50 NSCLC (stage I-IIIA) and in several normal lung samples all of which had previously been studied for cyclin D1 (DNA, RT-PCR, immunostaining), p16INK4a (DNA, RT-PCR, immunostaining), and pRb (immunostaining). As assessed by immunoblotting and immunostaining, p21 was expressed at low levels in normal lung tissue with immunoreactivity seen in a small number of bronchial epithelial cells only. In NSCLC, p21 expression (> or =10% of positive cells) was observed in 42% (21/50) of cases. High p21 expression was associated with well differentiated tumours (p = 0.01) and cyclin D1 nuclear staining (p = 0.02). Furthermore, we found an inverse correlation with p16INK4a (p = 0.004) and a direct correlation with pRb expression (p = 0.02). Risk of relapse was associated with p16INK4a and p21 status with no relapse in patients with normal p16INK4a and p21. Our results confirm that a large number of NSCLC have a low level of p21 expression. The associations of p21 and nuclear cyclin D1, pRb, p16INK4a support the relevance of pathways linked to lung carcinogenesis that involve p21 but may act in addition to direct CDK inhibition.     

Individuals with presumably hereditary uveal melanoma do not harbour germline mutations in the coding regions of either the P16INK4A, P14ARF or cdk4 genes

In familial cutaneous malignant melanoma (CMM), disruption of the retinoblastoma (pRB) pathway frequently occurs through inactivating mutations in the p16 (p16INK4A/CDKN2A/MTS1) gene or activating mutations in the G1-specific cyclin dependent kinase 4 gene (CDK4). Uveal malignant melanoma (UMM) also occurs in a familial setting, or sometimes in association with familial or sporadic CMM. Molecular studies of sporadic UMM have revealed somatic deletions covering the INK4A-ARF locus (encoding P16INK4A and P14ARF) in a large proportion of tumours. We hypothesized that germline mutations in the p16INK4A, p14ARF or CDK4 genes might contribute to some cases of familial UMM, or to some cases of UMM associated with another melanoma. Out of 155 patients treated at the Institut Curie for UMM between 1994 and 1997, and interviewed about their personal and familial history of melanoma, we identified seven patients with a relative affected with UMM (n = 6) or CMM (n = 1), and two patients who have had, in addition to UMM, a personal history of second melanoma, UMM (n = 1), or CMM (n = 1). We screened by polymerase chain reaction single-strand conformation polymorphism the entire coding sequence of the INK4A-ARF locus (exon 1alpha from p16INK4A, exon 1beta from p14ARF, and exons 2 and 3, common to both genes), as well as the exons 2, 5 and 8 of the CDK4 gene, coding for the functional domains involved in p16 and/or cyclin D1 binding. A previously reported polymorphism in exon 3 of the INK4A-ARF locus was found in one patient affected with bilateral UMM, but no germline mutations were detected, either in the p16INK4A, p14ARF or CDK4 genes. Our data support the involvement of other genes in predisposition to uveal melanoma.     

Loss of p16(INK4a) expression correlates with decreased survival in pediatric osteosarcomas

Abnormalities of the G1 cell-cycle checkpoint are commonly reported in cancers at various anatomic sites. pRB, p16(INK4a) and cyclin D1 are critical G1-checkpoint proteins responsible for maintaining the balance of cellular proliferation. We examined a series of 38 pediatric osteosarcomas for abnormal expression of pRB, p16(INK4a) and cyclin D1 by immunohistochemical analysis of archival biopsy specimens. Overall, 17/38 (45%) osteosarcomas showed evidence of G1-checkpoint abrogation, including 11/38 (29%) with loss of pRB expression and 6/38 (16%) with loss of p16(INK4a) expression. Cyclin D1 over-expression was not detected. There was an inverse correlation between loss of pRB and p16(INK4a) expression (p = 0.07). pRB and p16(INK4a) abnormalities were independent of site of disease, presence of metastasis at diagnosis and percentage of tumor necrosis in the resection specimen. Clinical follow-up was available on all patients (median 31.6 months, range 5.9-116 months). Absence of p16(INK4a) expression significantly correlated with decreased survival in univariate analysis (p = 0.03), while loss of pRB expression did not affect survival. Immunohistochemical analysis of p16(INK4a) expression in pediatric osteosarcomas may be a useful adjunctive marker of prognosis.     

Abrogation of the retinoblastoma tumor suppressor checkpoint during keratinocyte immortalization is not sufficient for induction of centrosome-mediated genomic instability

Deregulation of the retinoblastoma (pRB) tumor suppressor pathway and telomerase activation have been identified as rate-limiting steps for immortalization of primary human epithelial cells. However, additional molecular aberrations including p53 inactivation, ras activation, and deregulation of protein phosphatase 2A activity are necessary for full transformation of immortalized epithelial cells. Genomic instability is observed in most human tumors and constitutes an important mechanism to allow emerging tumor cells to acquire additional mutations to efficiently overcome selection barriers during carcinogenic progression. In an attempt to model oral cancer in a human cell-based system, we analyzed normal oral epithelial keratinocytes with the pRB pathway dysregulated by loss of expression of the cyclin-dependent kinase (cdk) 4/cdk6 inhibitor p16(INK4A) and/or ectopic expression of cdk4 or expression of the human papillomavirus (HPV) type 16 E7 oncoprotein. Ectopic expression of cdk4 and HPV-16 E7 was equally efficient in extending the life span of normal oral keratinocytes, and each was able to cooperate with telomerase (hTERT) to immortalize these cells. HPV-16 E7/hTERT-immortalized normal oral keratinocytes showed centrosome abnormalities, whereas populations of cdk4/hTERT-immortalized cells or hTERT-immortalized cells that had lost expression of p16INK4A showed no such abnormalities. These results demonstrate that disruption of the p16INK4A/pRB checkpoint of epithelial cell immortalization does not necessarily lead to centrosome-associated genomic instability.     

Disruption of the p16/cyclin D1/retinoblastoma protein pathway in the majority of human hepatocellular carcinomas.

p16, cyclin D1 and retinoblastoma protein (pRB) regulate G1 to S transition and are commonly targeted in various cancers. However, few studies have simultaneously examined all components of the p16/cyclin D1/pRB pathway (RB pathway) in hepatocellular carcinoma (HCC). To clarify the role of the disruption of the RB pathway in HCC, we analyzed p16, pRB and cyclin D1 in 47 HCCs. Inactivation of p16 was detected in 30 of 47 HCCs (64%) by Western blot analysis and significantly correlated with hypermethylation of the promoter of this gene. pRB expression was found to be absent in 13 of 47 HCCs (28%) by immunohistochemistry. We found that 38 of 47 HCCs (81%) contained at least one inactivation in either pRB or p16. Furthermore, there was a significant inverse correlation between p16 and pRB inactivation (p = 0.041). Overexpression of cyclin D1 was detected in 5 of 47 HCCs (11%) by immunohistochemistry. The cases with cyclin D1 overexpression exhibited an advanced clinicopathological appearance and also contained inactivation of pRB and/or p16. These findings suggest that inactivation of pRB and/or p16 is a major event in human hepatocarcinogenesis, while cyclin D1 overexpression may confer additional growth advantages to the tumor in addition to pRB and/or p16 inactivation in HCC.     

Identification of p18 INK4c as a tumor suppressor gene in glioblastoma multiforme

Genomic alterations leading to aberrant activation of cyclin/cyclin-dependent kinase (cdk) complexes drive the pathogenesis of many common human tumor types. In the case of glioblastoma multiforme (GBM), these alterations are most commonly due to homozygous deletion of p16(INK4a) and less commonly due to genomic amplifications of individual genes encoding cyclins or cdks. Here, we describe deletion of the p18(INK4c) cdk inhibitor as a novel genetic alteration driving the pathogenesis of GBM. Deletions of p18(INK4c) often occurred in tumors also harboring homozygous deletions of p16(INK4a). Expression of p18(INK4c) was completely absent in 43% of GBM primary tumors studied by immunohistochemistry. Lentiviral reconstitution of p18(INK4c) expression at physiologic levels in p18(INK4c)-deficient but not p18(INK4c)-proficient GBM cells led to senescence-like G(1) cell cycle arrest. These studies identify p18(INK4c) as a GBM tumor suppressor gene, revealing an additional mechanism leading to aberrant activation of cyclin/cdk complexes in this terrible malignancy.     

International cancer research grants

Ahderrant cyclin expression has been implicated in oncogenesis in a number of human cancers. Since alterehd function of regulators of cyclin-dependent kinase (CDK) activity other than cyclins, in particular CDK inhibitors, might play a similar role in oncogenesis, we examined the expression and regulation of the CDK inhibitors p16^INk4 p15^INK4B and p21^WAF1/CIP1 in human breast cancer cell lines. Both the INK4 and INK4B genes were homozygously deleted in 3 cell lines, while INK4 alone was deleted in 2 cell lines. A further 2 cell lines displayed loss of an allele at this locus, and in 1 of these the remaining allele contained a mis-sense mutation within the coding region of the p16^INK4 protein. The majority of cell lines examined, including 2 normal mammary epithelial cell strains, expressed low levels of INK4 mRNA and low or undetectable levels of INK4B mRNA. However, INK4 mRNA was expressed at high levels in 5 cell lines, and this was associated with deletion or inactivation of the retinoblastoma susceptibility gene product pRB but not with mutation of TP53. No deletions of the WAF1/CIP1 gene were observed, but WAF1/CIP1 mRNA levels were reduced in cell lines with TP53 mutation. Transfection of a p16^INK4 expression vector into MDA-MB-231 cells lacking the INK4 gene failed to produce any p16^INK4-expressing cell lines, suggesting that such cells were selected against in continuous culture. Despite the frequent deletion of INK4 in breast cancer cell lines, no evidence was obtained for INK4 deletions in DNA from 45 primary breast carcinomas. Thus, homozygous deletion of the INK4 gene appears to be a rare event in primary breast cancer.     

Alterations of pRb1-cyclin D1-cdk4/6-p16(INK4A) pathway in endometrial carcinogenesis

The retinoblastoma protein pathway (pRb1-cyclin D1-cdk4/6-p16(INK4A)) participates in the regulation of the cellular processes at the transition of G1/S phases of the cell-cycle. Derailments of this pathway, caused either by lack of pRb1 or p16(INK4A) expression or overexpression of cyclin D1 and/or cdk4/6, are implicated in the deregulation of the cell-cycle machinery, resulting in uncontrolled cell proliferation, tumor heterogeneity, invasion and metastasis. Several studies conducted so far have assessed the deregulation of the pRb1-pathway components in various human tumors and cell-lines, provided these pathway alterations play an obligatory role in tumorigenesis. This review briefly summarizes the current information on the pRb1-cyclin D1-cdk4/6-p16(INK4A) alterations in sporadic uterine cancer, placing emphasis on the influence on the dualistic model of endometrial carcinogenesis.