In an effort to discover genes that contribute to the pathogenesis of GBM, we used Affymetrix 250K Gene Chip Arrays to identify recurrent copy number alterations in a panel of 58 GBM tumor samples (). Focal deletions of the PTPRD
gene on chromosome 9p23-24.1 were among the most prevalent deletions detected, present in 14% of the GBM samples studied (; Supplementary Table S1
). This frequency of focal deletion is higher than that of PTEN (9%) and similar to that of CDKN2C
(also named p18INK4c
, 16%), a recently identified GBM tumor suppressor gene (14
). Larger-scale loss of the PTPRD
gene was present in an additional 33% of the samples (; Supplementary Table S2
). Intriguingly, several studies have suggested the presence of another important tumor suppressor gene on chromosome 9p telomeric to the CDKN2A/B locus in tumor types including astrocytoma, melanoma, and lung adenocarcinoma (16
). We therefore considered PTPRD
to be an attractive candidate as a GBM tumor suppressor gene, and possibly relevant to a range of other tumor types as well.
Figure 1 PTPRD is deleted at high frequency in GBM. A, most frequently deleted genes in 58 GBM tumor samples as determined by Affymetrix 250K SNP microarray analysis. B, copy number analysis of SNP microarray data shows focal (<10 Mb) deletions of chromosome (more ...)
To determine if PTPRD is genetically altered by mutation during GBM tumorigenesis, we sequenced the 35 coding exons of the PTPRD
gene in tumor samples lacking focal deletions of PTPRD and in corresponding normal tissue (when available). This sequence analysis identified somatic mutations of the PTPRD
gene in three samples, including two missense mutations and one nonsense mutation (; Supplementary Fig. S1
). Additionally, we identified a heterozygous germ-line mutation that was accompanied by somatic loss of the wild-type allele in the tumor of a GBM patient with a history of multiple primary malignancies (). This mutation is not a reported single nucleotide polymorphism (SNP) and was not present in any of >100 alleles of PTPRD sequenced during the course of this study. Together, these data show that PTPRD is altered by somatic mutation during GBM pathogenesis and raise the intriguing possibility that germ-line mutation of PTPRD might lead to a predisposition to the development of GBM and other tumor types.
Figure 2 Identification of somatic and inherited mutations of PTPRD in GBM. A, one nonsense and three missense mutations were identified in GBM samples. ^, genomic position is based on the hg18 genome assembly; #, transcript ENST00000381196 was used for annotation (more ...)
To determine if mutations of PTPRD were present in a second tumor type proposed to harbor an additional 9p tumor suppressor gene, we sequenced PTPRD in 57 melanoma tumor samples. Two somatic nonsense mutations and eight somatic missense mutations were identified (; Supplementary Figs. S2 and S3
) in a total of seven samples. All of these mutations were C/G>T/A transversions, consistent with UV-induced DNA damage. Additionally, three of the mutations were dinucleotide CC>TT mutations caused by the formation of UV-induced cyclobutane pyrimidine dimers (Supplementary Fig. S3
). Three of the seven samples harboring somatic mutations of PTPRD displayed loss of heterozygosity (LOH) of the wild-type allele. Furthermore, tumor 76T harbored four independent mutations of the gene, increasing the likelihood that both alleles of the gene had been targeted by mutation in this sample. Interestingly, five of these seven samples with PTPRD mutation also harbor activating mutations of either B-Raf or N-Ras (data not shown). This 12% mutation frequency makes PTPRD
among the most commonly mutated genes in sporadic melanoma reported to date, which include B-Raf
(0–5%), and PIK3CA
(<1%; refs. 21
Identification of somatic mutations of PTPRD in malignant melanoma
The 14 mutations of PTPRD reported here are distributed roughly evenly throughout the various extracellular and intracellular domains of the encoded PTPRD protein (), although there seems to be a mini-hotspot in the first and second fibronectin type III repeat.
Figure 3 Schematic of the PTPRD protein and the location of all mutations reported to date in human cancer. The mutations in colon cancer and lung cancer were previously reported in refs. 11 and 12. S, signal peptide; Ig, immunoglobulin-like C2-type domain; FN (more ...)
Despite its potential importance, functional data implicating PTPRD deletion or mutation in tumorigenesis are lacking. To determine if PTPRD has the growth-suppressing properties expected of a broad-spectrum tumor suppressor gene, we examined the functional consequences of reconstituting PTPRD expression in GBM and melanoma cells. A 5.1-kb human PTPRD cDNA was obtained, cloned into a lentiviral expression vector, and packaged into infectious lentivirus as described in Materials and Methods. Infection of H4 cells that harbor biallelic deletion of PTPRD (; Supplementary Table S1
) with lenti-PTPRD led to expression of both the PTPRD proprotein and its mature cleavage products (which then reassemble at the cell membrane to form a heterodimer; ; ref. 23
). Infection of H4 cells with lenti-PTPRD but not vector alone led to a transient growth arrest evidenced by a reduction in BrdUrd incorporation and an increase in both G1
cell populations (). Infection with lenti-PTPRD had a similar effect on 8MGBA cells, which also harbor a focal deletion of PTPRD (; data not shown).
Figure 4 Expression of PTPRD in GBM and melanoma cells harboring deletions and mutations causes growth suppression and apoptosis. A, Western blot for PTPRD shows reconstitution of PTPRD expression in 8MGBA and H4 GBM cells infected with lenti-PTPRD. Both cell (more ...)
We next infected two primary melanoma cell cultures harboring homozygous missense mutations of PTPRD [16T and 86T with G446E in the second fibronectin type III domain and V1565I in the first PTP catalytic (PTPc) domain, respectively]. Infection of both primary cell cultures with wild-type PTPRD but not vector alone led to significant growth inhibition and decrease in cell viability (), as well as a substantial, time-dependent increase in apoptotic cells (). These are the first reported data indicating that PTPRD has growth-suppressive properties when expressed in human cancer cells, supporting the hypothesis that PTPRD is a bona fide human tumor suppressor gene.
We next sought to examine the consequences of tumor-derived mutations on PTPRD function in these assays. To do this, five tumor-derived mutations were introduced into lenti-PTPRD as described in Materials and Methods, including two mutations in the second fibronectin type III domain mini-hotspot (one each from GBM and melanoma), one mutation in the first PTPc domain (melanoma), and two mutations flanking the proprotein cleavage site (one each from GBM and melanoma). Initially, H4 cells were infected with wild-type and mutant lenti-PTPRD, protein lysates were prepared, and PTPRD expression was documented by Western blot. As shown in , infection of H4 cells with lentivirus expressing either wild-type or mutant PTPRD resulted in similar levels of protein expression. However, there was a marked decrease in growth inhibition as measured by BrdUrd incorporation, indicating that each of the five tumor-derived mutants alleviated the growth suppression activity of PTPRD, albeit to differing extents (). Next, 16T melanoma cells were similarly infected with wild-type and mutant PTPRD lentiviruses. As depicted in , wild-type PTPRD led to apoptosis of ~75% of the cells at 10 days after infection, whereas the mutant PTPRD lentiviruses led to a substantially reduced fraction of cells that had undergone programmed cell death. When taken together, these experiments show that tumor-derived mutations of PTPRD attenuate its function, confirming that the mutations of PTPRD are likely to be pathogenic.
Figure 5 Tumor-derived mutations compromise the growth-suppressive function of PTPRD in GBM and melanoma cells. Five tumor-derived mutations were introduced into lenti-PTPRD. A, Western blot analysis for PTPRD protein shows equivalent expression of wild-type and (more ...)