Evidence of ultraviolet type mutations in xeroderma pigmentosum melanomas

Abstract

To look for a direct role of ultraviolet radiation (UV) exposure in cutaneous melanoma induction, we studied xeroderma pigmentosum (XP) patients who have defective DNA repair resulting in a 1000-fold increase in melanoma risk. These XP melanomas have the same anatomic distribution as melanomas in the general population. We analyzed laser capture microdissection samples of skin melanomas from XP patients studied at the National Institutes of Health. The tumor suppressor gene PTEN was sequenced and analyzed for UV-induced mutations. Samples from 59 melanomas (47 melanomas in situ and 12 invasive melanomas) from 8 XP patients showed mutations in the PTEN tumor suppressor gene in 56% of the melanomas. Further, 91% of the melanomas with mutations had 1 to 4 UV type base substitution mutations (occurring at adjacent pyrimidines) (P < 0.0001 compared to random mutations). We found a high frequency of amino-acid-altering mutations in the melanomas and demonstrated that these mutations impaired PTEN function; UV damage plays a direct role in induction of mutations and in inactivation of the PTEN gene in XP melanomas including in situ, the earliest stage of melanoma. This gene is known to be a key regulator of carcinogenesis and therefore these data provide solid mechanistic support for UV protection for prevention of melanoma.

Fig. 1.

Melanoma identification and laser capture microdissection of melanoma cells. (A) A pigmented lesion on the left upper arm of a 52-year-old XP patient, XP295BE, showing the melanoma features of asymmetry, border irregularity, color variation, and large diameter. (B) Dermatoscopic image of A (20× magnified) showing a central, asymmetric, homogeneously hyperpigmented area with irregularly distributed black dots (arrows) and irregular streaks (arrowheads). (C) Atypical melanocytes are present singly and in nests at the dermal-epidermal junction (arrows) and show focal extension into the superficial dermis (hematoxyalin and eosin stain). (D) Melan-A staining showing melanoma cells expressing Melan-A (arrows). (E) Tissue remaining after capture of the melanoma cells showing high efficiency of capture (arrows). (F) Nests of melanoma cells captured (arrows) along with small amount of adjacent tissue.

Fig. 2.

Location of PTEN mutations in melanomas and effect of mutations on PTEN function. (A) Sites of 52 PTEN cDNA base substitution mutations found in 33 melanomas from 8 XP patients. The 9 exons of the 1212 bp PTEN cDNA are indicated. Mutated bases are numbered. UV type mutations are indicated above the exons and non-UV type mutations are indicated below the exons. An * indicates cancer-associated mutation listed in the Sanger COSMIC database (22). Vertical arrows indicate mutations tested for alteration of PTEN function. Arrows with X indicate mutations that result in decreased PTEN function. (B) Sites of 33 PTEN nonsynonymous amino acid substitution mutations and 2 nonsense mutations found in 33 melanomas from 8 XP patients. The 404 aa protein has a dual specificity protein phosphatase domain from amino acid 25 to 179, a tyrosine specific protein phosphatase region from amino acid 123 to 134 and a C2 calcium/lipid-binding region, (CaLB) from amino acid 190 to 347. The altered amino acids are numbered. UV type mutations are indicated above the sequence and non-UV type mutations are indicated below. An * indicates cancer associated mutation listed in Sanger COSMIC database (22). Vertical arrows indicate mutations tested for alteration of PTEN function. Arrows with X indicate mutations that result in decreased PTEN function. (C) Functional assay of phosphorylation of Akt by selected PTEN mutants. NCI-H1155 PTEN-null cells were transfected with pCMV5 HA-PKB/Akt (containing hemagglutinin (HA) tagged PKB/Akt) plus pCMV5 with wild type (w.t.) (lanes 2 and 8) or mutated (lanes 3–6, 9–10) PTEN or empty vector (mock) (lanes 1 and 7). After 24 h, the cells were lysed and immunoprecipitated with anti-HA antibodies. This HA-PKB/Akt was analyzed by Western blotting with an anti-Akt phosphoserine 473 antibody (Middle). The membrane was then stripped and analyzed using an antibody against total Akt (Lower). Expression of transfected PTEN proteins was confirmed by anti-PTEN Western blot analysis of cell lysates (Upper). The phosphorylation of Akt by mutants p.F154L (lane 3), p.L325F (lane 4), and p.P95S (lane 5) indicates loss of PTEN suppressor function while the low level of phosphorylation of Akt by mutants p.Q110R (lane 6), p.D301N (lane 9), and p.S362L (lane 10) indicates preservation of PTEN suppressor function. (D) Relationship of number of PTEN base substitution mutations per melanoma to melanoma frequency (1- cumulative frequency). The data are consistent with a random (Poisson) distribution of mutations.