DICER1 Mutations in Familial Pleuropulmonary Blastoma

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Science  21 Aug 2009:
Vol. 325, Issue 5943, pp. 965
DOI: 10.1126/science.1174334


Pleuropulmonary blastoma (PPB) is a rare pediatric lung tumor that is often part of an inherited cancer syndrome. PPBs consist of mesenchymal cells that are susceptible to malignant transformation and cysts lined by epithelial cells. In a subset of patients, overgrowth of the cysts by mesenchymal cells leads to sarcoma formation. Here, we show that 11 multiplex PPB families harbor heterozygous germline mutations in DICER1, a gene encoding an endoribonuclease critical to the generation of small noncoding regulatory RNAs. Expression of DICER1 protein was undetectable in the epithelial component of PPB tumors but was retained in the malignant mesenchyme (sarcoma). We hypothesize that loss of DICER1 in the epithelium of the developing lung alters the regulation of diffusible factors that promote mesenchymal proliferation.

Pleuropulmonary blastoma (PPB) is a rare pediatric tumor of the lung that arises during fetal lung development and is often part of an inherited cancer syndrome [Online Mendelian Inheritance in Man (OMIM) 601200]. PPBs contain both epithelial and mesenchymal cells. Early in tumorigenesis, cysts form in lung airspaces, and these cysts are lined with benign-appearing epithelium. Mesenchymal cells susceptible to malignant transformation reside within the cyst walls and form a dense “cambium” layer beneath the epithelial lining. In a subset of patients, overgrowth of the mesenchymal cells produces a sarcoma, a transition that is associated with a poorer prognosis (1). The natural history of PPBs is consistent with a multistep genetic model of tumor progression (2).

About 20% of children with PPB have a family history of neoplasia, especially cystic nephroma of the kidney and rhabdomyosarcoma (3). We mapped the PPB locus to chromosome 14q with a family-based linkage study on four families with inherited predisposition to PPB (4) (figs. S1 and S2). Of 72 genes within the 7-Mb region of interest, DICER1 was an attractive candidate because of its role in lung development. DICER1 encodes a ~218-kd ribonuclease (RNase) III endonuclease that participates in the generation of small RNAs [microRNAs (miRNAs) and small interfering RNAs (siRNAs)] (5). miRNAs negatively regulate gene expression and have been implicated in multiple biological processes including stem cell maintenance, organogenesis, and oncogenesis. Conditional loss of Dicer1 in the developing mouse lung results in cystic airways, disruption of branching morphogenesis, and mesenchymal expansion that resembles the early stage of PPB (6).

We identified heterozygous germline mutations in DICER1 by sequencing genomic DNA from affected members in each of 11 families (four included in the linkage study and seven additional families) (Fig. 1A, fig. S3, and table S1). In 10 of these families, the mutations result in proteins truncated proximal to the two carboxy-terminal RNase III functional domains in DICER1 (Fig. 1B) and thus likely cause loss of function. The missense mutation [Leu1583→Arg1583 (L1583R)] detected in the 11th family (family C) affects an evolutionarily conserved amino acid, and the nonpolar to polar change was neither a previously reported sequence variant [National Center for Biotechnology Information Single Nucleotide Polymorphism (NCBI SNP) database Build 128], nor was it detected in 360 cancer-free controls tested by Pyrosequencing (QIAGEN, Incorporated, Valencia, CA).

Fig. 1

Germline DICER1 mutations and DICER1 protein expression in PPB. (A) DICER1 sequence alteration in one of the families in the linkage study. (B) Schematic of DICER1 protein showing the locations of DICER1 mutations in the 11 PPB families. Vertical dotted lines with arrows indicate truncating mutations or insertions or deletions in 10 families, all occurring proximal to the RNase III domains (yellow). The larger arrow with the solid line marks the missense mutation, which occurs between the RNase III domains. (C) DICER1 protein is not detectable in benign-appearing tumor-associated epithelium (arrows) but is present in the mesenchymal tumor cells forming the underlying cambium layer (anti-DICER1 with brown chromagen and hematoxylin counterstain; original magnification 400×).

Apart from PPB-associated tumors in a subset of family members, the majority of obligate carriers with DICER1 mutations are phenotypically normal, suggesting that loss of one DICER1 allele is compatible with normal development and insufficient for tumor formation. Mice haploinsufficient for Dicer1 also show no overt phenotypic abnormalities (7). DICER1 immunohistochemistry of PPB tumors suggests expression from the wild-type allele is lost in tumor-associated epithelium in six of the seven families harboring PPBs with a residual epithelial cystic component but is retained in the mesenchymal tumor cells (Fig. 1C and fig. S5). DICER1 is normally present in lung bronchial and alveolar epithelium throughout life. The areas of absent staining in the tumor epithelium were segmental in most cases but were clearly evident in areas overlying cambium layers. The genetic basis for this altered expression in epithelium is unknown, but the phenotype recapitulates that seen in the Dicer1−/− mice. Interestingly, the tumor-associated epithelial cells lacking DICER1 expression appeared to be normally differentiated.

On the basis of the loss of DICER1 protein expression specifically in lung epithelium overlying PPB, we hypothesize that PPB may arise through a novel mechanism of non–cell-autonomous cancer initiation. Conceivably, loss of DICER1 in developing lung epithelium alters miRNA-dependent regulation of diffusible growth factors that promote mesenchymal cell proliferation. Whether and how DICER1 haploinsufficiency in the mesenchymal cells contributes to pathogenesis remain open questions.

Supporting Online Material

Materials and Methods

SOM Text

Figs. S1 to S5

Tables S1 to S5


References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. The authors (D.A.H., P.J.G., J.R.P., and Y.M.) have filed a patent related to this work.
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