The absence of correlation with p16 protein expression and angiogenesis suggests that other regulatory pathways and mechanisms might be influenced by ID1 in melanomas.
Here we examined the ability of p16-derived peptides to mimic p16 function in two exemplary human melanoma cell lines: the p16-defective, pRb-positive A375M cells and p16-positive, pRb-defective A2058 cells.
We found that p16 expression was inversely correlated with tumor progression and was significantly lower in melanomas, including in situ lesions, than in nevi.
The p16 protein was weakly expressed in one of the metastatic melanoma cell lines (FM55M1) and negative in the other metastasis (FM55M2) as compared to their matched primary melanoma cells (FM55P).
A cytostatic effect of flavopiridol on the growth of six melanoma cell lines with a mutated or non-expressed p16 (p16-) was seen at low concentrations of flavopiridol (mean 50% inhibitory concentration [IC(50)] = 12.5 nM), while the three melanoma cell lines with intact p16 (p16+) required higher concentrations (mean IC(50) = 25 nM) to produce this effect.
Taken together, these findings are consistent with loss of p16 being a late event in the progression of sporadic primary melanomas, being associated with tumours of a more aggressive nature.
This partial functional defect may complement the clearly defective p16 del (62-69) mutant and thus contribute to melanoma development in patients carrying the 24bp deletion in CDKN2A.
These studies reveal that LOH and homozygous deletion can affect 9p21 and the p16 locus early in putative precursor lesions of melanoma, even prior to the establishment of cytologically evident aberrant histology.
Germline mutations of the cell-cycle regulator p16 (also called "CDKN2A") in kindreds with melanoma implicate this gene in susceptibility to malignant melanoma.
At present, the most useful methods of risk assessment are those performed on the following genes: BRCA1 and BRCA2 especially for hereditary breast and ovarian cancer, hMLH1 and hMSH2 for hereditary non polyposis colorectal cancer, APC for familial adenomatous polyposis, ret for medullary thyroid carcinoma, p53 for the Li-Fraumeni syndrome, p16 for melanoma and RB1 for retinoblastoma.
These data suggest a role for Id1 in regulating p16/Ink4a expression in early melanomas and demonstrate that later genetic changes may provide for irreversible loss of p16 expression in advanced stages of this tumor.
We sequenced 1,327 base pairs (bp) of CDKN2A, making up 1,116 bp of the 5' UTR and promoter, all of exon 1, and 61 bp of intron 1, in at least one melanoma case from 110 Australian families with three or more affected members known not to carry mutations within the p16 coding region.
Two p16 germline mutations were identified: G101W, which has been previously observed in a number of melanoma kindreds, and G122V, a novel missense mutation.
These recent advances open up the possibility of genetic testing for melanoma susceptibility in the setting of familial melanoma and suggest novel therapeutic strategies for melanoma based on gene therapy or small molecule mimicry targeted to the correction of defects in the p16 regulatory pathway.(J Am Acad Dermatol 2000;42:705-22.)
Thus the addition of activating ras mutations to a melanoma cell line already deficient in p16 leads to enhanced proliferation, survival and migration in vitro and to enhanced subcutaneous tumour formation in vivo.
Germline mutations in BRCA2 have been shown to predispose to both breast and pancreatic cancer, germline mutations in p16 to melanoma and pancreatic cancer (the FAMMM syndrome), and genetic mutations in STK11/LKB1 to pancreatic cancer in patients with the Peutz-Jeghers Syndrome (PJS).
This paper reviews the current literature on p16 expression in melanoma and pancreatic cancer, explores factors that place patients with these cancers in categories of high risk for metastases or recurrence, and addresses whether aberrant gene expressions should influence awareness of and current recommendations for the management of these aggressive cancers.
We have used a melanoma cell line (MM96L) with no functional p16, as the basis for a "semi-in vivo" transfection-based assay for exogenous p16 functionality based on the growth parameters of the cells and the behaviour of variant proteins after transfection of different CDKN2A cDNAs.