Alterations of the p16-pRb Pathway and the Chromosome Locus 9p21–22 in Non-Small-Cell Lung Carcinomas

Abstract

The p16-pRb and p53-MDM2 pathways represent vital cell cycle checkpoints. Recent studies provide evidence that these pathways are directly linked via MDM2-pRb interaction and p53 suppression of the RB1 gene. In the present study we investigated the alterations of this G1 phase protein network using immunohistochemical and molecular methods in a series of 68 non-small-cell lung carcinomas (NSCLCs) and correlated the findings with clinicopathological features and prognosis of the patients. Aberrant expression (Ab) of p16 and pRb was observed in 33 (49%) and 27 (40%) of the carcinomas, respectively. Analysis of the region that encodes for p16 by deletion mapping, a polymerase chain reaction (PCR)-based methylation assay and PCR single-strand conformation polymorphism (SSCP) analysis revealed that deletions and transcriptional silencing by methylation might represent the main mechanisms of CDKN2/p16ink4a inactivation in NSCLCs. The results of deletion mapping also suggest that other tumor suppressor genes may reside at the 9p21-22 region, which encodes for CDKN2/MTS1/p16ink4a, p14ARF, and MTS2/p15ink4b. In addition, microsatellite instability was observed with a frequency of 16% in the 9p21-22 chromosome area. Overexpression (P) of p53 and MDM2 proteins was found in 39 (58%) and 47 (70%) of the cases, respectively. A highly significant association was observed between p53 overexpression and p53 mutations (P = 0.006). Statistical analysis of the expression patterns of the biologically relevant molecules (p16/pRb, p53/MDM2, MDM2/pRb, and p53/pRb) showed coincident overexpression of p53 and MDM2 (P = 0.04) and that abnormal pRb was correlated with elevated levels of MDM2 (P = 0.013) and p53 (P = 0.01), respectively. We suggest that deregulated expression of these molecules may act synergistically. An important finding of the study was that multiple impairments (three and four molecules affected) of the p16/pRb/p53/MDM2 network occurred in a large proportion (43%) of the carcinomas. This finding in addition to the absence of correlation with clinical stage of the tumors suggests that multiple hits of this network may be a relatively early event in the development of a subset of NSCLCs. The relationship between the factors examined in the present study, clinicopathological features, and survival of the patients did not reveal any significant correlations with the exception of smoking, which was associated with microsatellite alterations (loss of heterozygosity and microsatellite instability) at the 9p21-22 locus (P = 0.04) and the immunophenotypes p53(P)/MDM2(P) (P = 0.04) and p16(Ab)/pRb(Ab)/p53(P)/MDM2(P) (P = 0.03), respectively. We suggest that in a subset of NSCLCs, simultaneous deregulation of the members of this network may represent one way of initiating the oncogenic procedure whereas in other NSCLC subgroups alternative pathways may play this role.

p16Ink4a overexpression in cancer: a tumor suppressor gene associated with senescence and high-grade tumors

“…In the same samples, high p16 Ink4a protein expression was observed, and there was a statistical correlation between Rb loss of heterozygosity and p16 Ink4a expression levels (P ¼ 0.01). Actually, an inverse relationship between p16 Ink4a and Rb expression has been reported in breast cancer (Dublin et al, 1998;Gorgoulis et al, 1998), and recently in lung cancer (Bastide et al, 2009). Moreover, studies by our group have provided evidence of p16 Ink4a overexpression in a subgroup of undifferentiated high-grade pleomorphic sarcomas (Figure 2), and Rb loss of heterozygosity was found in 5/6 of these cases (unpublished data).…”

Overall Survival in Aggressive B-Cell Lymphomas Is Dependent on the Accumulation of Alterations in p53, p16, and p27

“…53,54 Several in vitro studies have provided evidence of the synergy between p53 and p16, and concurrent alterations of p16 and p53 have been shown to confer a higher aggressiveness on different tumor types. 35,38 This series analyzing LBCLs shows that the concurrent inactivation of these CKIs [p21 (provided by p53 mutations), p16, and p27], operating in different but co-operative pathways has an accumulative effect on overall survival of LBCL patients (P ϭ 0.0218). Whether this phenomenon is valid only in LBCLs or could be extrapolated to other tumors is a debatable point.…”

Downregulation of the KIP family members p27KIP1 and p57KIP2 by SKP2 and the role of methylation in p57KIP2 inactivation in nonsmall cell lung cancer

“…These were performed according to protocols previously described. 36,43 The primers and thermal profiles used are given in Table II. LOH analysis. (i) Conventional LOH Analysis: PCR products were electrophoretically separated on 10% polyacrylamide gels and stained with silver nitrate as previously reported.…”

“…(i) Conventional LOH Analysis: PCR products were electrophoretically separated on 10% polyacrylamide gels and stained with silver nitrate as previously reported. 43 (ii) Capil- lary Electrophoresis: Heterozygous samples were subsequently analyzed by capillary chip electrophoresis on a 2100 Bioanalyser (Agilent). Evaluation.…”