Mechanism of Retinoblastoma Gene Inactivation in the Spectrum of Neuroendocrine Lung Tumors

Abstract

The retinoblastoma (RB) gene plays a key role in cell cycle control by regulation of G1 growth arrest. This gene is inactivated in some human cancers and in most small-cell lung carcinoma (SCLC) cell lines. The aim of this study was to analyze the mechanisms of RB silencing in freshly excised neuroendocrine (NE) tumors embracing the entire spectrum of NE lung neoplasms (typical and atypical carcinoids, large-cell neuroendocrine carcinomas [LCNECs], and SCLCs). To study the role and mechanism of RB inactivation in tumor differentiation and malignant potential, the status of the Rb protein was analyzed in 37 NE lung tumors, using immunohistochemistry with five Rb antibodies. Loss or altered expression of Rb protein was more frequently observed in high-grade NE lung carcinoma (23 of 28, 82%) than in typical and atypical carcinoids (1 of 9, 11%) (P < 0.001). Of 24 tumors with abnormal Rb staining, Southern blotting showed 1 to have undergone rearrangement, SSCP (single-strand conformation polymorphism) and sequencing showed that 6 (25%) exhibited mutations in exons 13-18 or 20-24 of the RB gene, and RT-PCR (reverse transcriptase-polymerase chain reaction) revealed that 14 (58%) showed a low level of or entirely absent RB mRNA (messenger RNA) expression, whereas hypermethylation of the CpG-rich island at the 5' end of the RB gene was not observed. Abnormal Rb protein expression was always associated with one of these three alternative mechanisms in the SCLCs analyzed, but in only 50% of LCNECs. These results indicate that inactivation of the RB gene is highly frequent in freshly excised high-grade NE lung tumors through distinct mechanisms including point mutations and frequent abnormal mRNA expression. Different modes of RB inactivation seem to be implicated along the spectrum of NE lung carcinomas, depending on differentiation state, phenotype, and malignancy grade.

Divergent cyclin B1 expression and Rb/p16/cyclin D1 pathway aberrations among pulmonary neuroendocrine tumors

“…21,23 Repeated studies have shown that carcinoid tumors were genetically divergent from high-grade large-cell neuroendocrine carcinoma and small-cell carcinoma, and had no, or only occasional, genetic events such as p53 mutation and Rb inactivation. 13,14,[24][25][26][27][28] In the present study, we demonstrated for the first time that cyclin B1 overexpression was also a very frequent event in high-grade neuroendocrine tumors and was found in most (84%) large-cell neuroendocrine carcinomas and small-cell carcinomas, while no typical carcinoid and only one (20%) atypical carcinoid overexpressed cyclin B1, indicating that regulation of cyclin B1 expression and G2/M arrest are consistently compromised in high-grade pulmonary neuroendocrine tumors, but are intact in typical carcinoid or only occasionally compromised in atypical carcinoid. The high frequency of cyclin B1 dysregulation in both the large-cell neuroendocrine carcinoma and small-cell carcinoma may reflect a similar alteration in cell cycle regulation at the G2/M transition, despite their distinct morphologies.…”

“…13,14,25,[27][28][29] On the other hand, divergence in chromosomal aberrations was also observed between them, 30 and the specific genetic alterations for either large-cell neuroendocrine carcinoma or small-cell carcinoma remain to be further characterized. In the present study, both large-cell neuroendocrine carcinoma and small-cell carcinoma showed almost equally frequent Rb pathway aberration.…”

“…8,9 However, to the best of our knowledge, no data concerning cyclin B1 expression status within the context of pulmonary neuroendocrine tumors are currently available. Furthermore, although overexpression of cyclin D1 and loss of Rb or p16 expression were also reported in various solid tumors, and cyclin D1 overexpression was reported to be of prognostic importance in some tumors, [10][11][12][13][14][15][16][17][18][19] aberrations in the Rb/p16/cyclin D1 pathway (Rb pathway) are not yet well characterized in pulmonary neuroendocrine tumors. 20 Thus, the present study was carried out to analyze cyclin B1 and D1 expression status, as well as aberrations in the Rb pathway, and their relationship to tumor proliferation activity and clinical outcomes in a spectrum of pulmonary neuroendocrine tumors.…”

Distinct pattern of E2F1 expression in human lung tumours: E2F1 is upregulated in small cell lung carcinoma

“…Relationships between E2F1, p53, pRb and hp14 ARF status in lung carcinoma Status of p53, pRb and hp14 ARF had been previously analysed in this series of tumours (Brambilla et al, 1993;Gazzeri et al, 1994Gazzeri et al, , 1998aGouyer et al, 1998). In these tumours, pRb function was inactivated, either by loss-of-function mutations or p16 INK4a loss and/or cyclin D1 overexpression in 29/36 (80%) NE tumours and 18/22 (82%) NSCLC respectively.…”

“…pRb pathway is di erentially disrupted in NSCLC and high grade NE tumours. Whereas loss of pRb protein expression (through mutations and/or loss of transcription) occurs in 85% of SCLC and LCNEC (Gouyer et al, 1998), pRb function is abolished in 85% of NSCLC either through loss of p16 INK4a protein expression (Xu et al, 1996;Gazzeri et al, 1998a), and/ or cyclin D1 overexpression (Brambilla et al, 1997). As E2F1 is one key component of these two pathways (O'Connor et al, 1995;Bates et al, 1998;Kowalik et al, 1998), any alterations a ecting them, especially pRb inactivation, are thought to deregulate its activity.…”