Activating point mutations in cyclin-dependent kinase 4 are not seen in sporadic pituitary adenomas, insulinomas or Leydig cell tumours

Abstract

Cell cycle dysregulation is one of the defining features of cancer. Cyclin-dependent kinase 4 (CDK4), together with its regulatory subunit cyclin D, governs cell cycle progression through the G1 phase. Cyclin-dependent kinase inhibitors, including p16 INK4A (encoded by CDKN2A), in turn regulate CDK4. In particular, dysregulation of the p16/CDK4/cyclin D complex has been established in a variety of types of human tumours. Dominant activating mutations affecting codon 24 of the CDK4 gene (replacement of Arg24 by Cys or His) render CDK4 insensitive to p16 INK4 inhibition and are responsible for melanoma susceptibility in some kindreds. However, 'knock-in' mice homozygous for the CDK4 R24C mutation were noted to develop multiple neoplasia, most commonly including endocrine tumours: pituitary adenomas, insulinomas and Leydig cell testicular tumours. We therefore speculated that sporadic human endocrine tumours might also harbour such mutations. The aim of the current study was to analyze the CDK4 gene for the two characterized activating mutations, R24C and R24H, in sporadic human pituitary adenomas, insulinomas and Leydig cell tumours. We used DNA extracted from 61 pituitary adenomas, and paired tumorous and neighboring normal genomic DNA extracted from 14 insulinoma and 6 Leydig cell tumour samples. Genomic DNA from patients with familial melanoma harbouring the R24C or the R24H mutations served as positive controls. All samples were subjected to PCR, mutation-specific restriction digests and/or sequencing. Both methodologies failed to detect mutations at these two sites in any of the sporadic endocrine tumours including pituitary adenomas, benign or malignant insulinomas or Leydig cell tumours, while the positive controls showed the expected heterozygote patterns. Protein expression of CDK4 was demonstrated by immunohistochemistry and Western blotting in pituitary and pancreatic samples. These data suggest that the changes in the regulatory 'hot-spot' on the CDK4 gene, causing various endocrine tumours in CDK4 R24C/R24C mice, are not a major factor in sporadic pituitary, insulin -cell or Leydig cell tumorigenesis.

Hotspot mutations in common oncogenes are infrequent in nasopharyngeal carcinoma

“…Cyclin-dependent kinase 4 (CDK4) is the chief catalytic subunit of the regulatory cyclin D that governs G1-to-S phase cell cycle progression (50). Dominant activating mutations affecting codon 24 of the CDK4 gene (R24H or R24C) render CDK4 insensitive to p16INK4 inhibition and are responsible for multiple neoplasia developing (50,51). Collectively, these data indicated that CDK4 mutation may play a role in oncogenesis in a subset of NPC patients.…”

Leydig-Cell Tumour in Children: Variable Clinical Presentation, Diagnostic Features, Follow-Up and Genetic Analysis of Four Cases

“…gip2 , and CDK4 genes, as a possible explanation for the phenotypical variability. However, no activating mutation was found in these genes previously reported to be involved in the pathogenesis of Leydig-cell tumours [3,4,5,6,7,8,9,10,11]. …”

“…Further, testes sparing enucleation of a Leydig-cell tumour seems to be sufficient in childhood, but the boy’s scrotum and further development needs ongoing follow-up as rarely a malignant process may occur in the future [23]. In addition, considering the previously reported mutations found in a few of Leydig-cell tumours [3,4,5,6,7,8,9,10,11], the absence of any activating mutations in LHR , as well as in both the ‘hot spot’ regions within the G-alpha subunits ( gsp and gip2 ) and in the regulatory ‘hot spot’ of the CDK4 genes in our patients, indicates molecular heterogeneity among Leydig-cell tumours, corresponding to its highly variable phenotype, which has not been reported previously.…”

“…Applying the conditions and the primers reported, PCR was performed to amplify the individual gene and the specifically activating regions of the LHR [7], gsp [9,16,17,18,19], gip2 [17,18,19], and CD4K [10] genes. Genomic DNA was amplified in a 50 µl PCR mix containing 220 µ M of each dNTPs, 66 m M Tris-SO 4 (pH 9.1), 19.8 m M (NH 4 )SO 4 , 2.2 m M MgSO 4 , 22 U/ml DNA polymerase and 30 pmol of each primer.…”

“…These include missense mutations in exons 8 and 9 of G s alpha (gsp) and exons 5 and 6 of G i alpha (gip2) gene [9]. In addition,cyclin-dependent kinase 4 (CDK4) governs cell cycle progression through G1 phase and deregulation of this kinase has been shown in a variety of human tumours [10]. Knock-in mice homozygous for the R24C CDK4 gene mutation were noted to develop several endocrine tumours including Leydig-cell tumour [11].…”