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, 10 (3), e1004216
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CDKN2D-WDFY2 Is a Cancer-Specific Fusion Gene Recurrent in High-Grade Serous Ovarian Carcinoma

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CDKN2D-WDFY2 Is a Cancer-Specific Fusion Gene Recurrent in High-Grade Serous Ovarian Carcinoma

Kalpana Kannan et al. PLoS Genet.

Abstract

Ovarian cancer is the fifth leading cause of cancer death in women. Almost 70% of ovarian cancer deaths are due to the high-grade serous subtype, which is typically detected only after it has metastasized. Characterization of high-grade serous cancer is further complicated by the significant heterogeneity and genome instability displayed by this cancer. Other than mutations in TP53, which is common to many cancers, highly recurrent recombinant events specific to this cancer have yet to be identified. Using high-throughput transcriptome sequencing of seven patient samples combined with experimental validation at DNA, RNA and protein levels, we identified a cancer-specific and inter-chromosomal fusion gene CDKN2D-WDFY2 that occurs at a frequency of 20% among sixty high-grade serous cancer samples but is absent in non-cancerous ovary and fallopian tube samples. This is the most frequent recombinant event identified so far in high-grade serous cancer implying a major cellular lineage in this highly heterogeneous cancer. In addition, the same fusion transcript was also detected in OV-90, an established high-grade serous type cell line. The genomic breakpoint was identified in intron 1 of CDKN2D and intron 2 of WDFY2 in patient tumor, providing direct evidence that this is a fusion gene. The parental gene, CDKN2D, is a cell-cycle modulator that is also involved in DNA repair, while WDFY2 is known to modulate AKT interactions with its substrates. Transfection of cloned fusion construct led to loss of wildtype CDKN2D and wildtype WDFY2 protein expression, and a gain of a short WDFY2 protein isoform that is presumably under the control of the CDKN2D promoter. The expression of short WDFY2 protein in transfected cells appears to alter the PI3K/AKT pathway that is known to play a role in oncogenesis. CDKN2D-WDFY2 fusion could be an important molecular signature for understanding and classifying sub-lineages among heterogeneous high-grade serous ovarian carcinomas.

Conflict of interest statement

Baylor College of Medicine has filed a provisional patent application based on the results of this report.

Figures

Figure 1
Figure 1. CDKN2D-WDFY2 is a highly frequent fusion transcript in HG-SC cancer samples and cell line.
(A) The results of nested RT-PCR for CDKN2D-WDFY2 in 60 HG-SC samples (denoted by “S”), 10 non-cancerous donor ovary samples (“OV”), and 4 non-cancerous donor fallopian tube (“FT”) samples are shown. NTC refers to “no template control”. S5 is the sample in which the fusion transcript was initially identified and this serves as the positive control. (B) The results of RT-PCR for CDKN2D-WDFY2 in five HG-SC cell lines (CaOV3, OV90, OVCAR8, OVCAR5 and OVCAR3), in addition to two endometrioid type cell lines (TOV112D and MDAH 2774) are shown.
Figure 2
Figure 2. CDKN2D-WDFY2 fusion transcript results from a genomic rearrangement of chromosome 19 and 13.
(A) CDKN2D gene is present on chromosome 19 and contains two exons while WDFY2 is present on chromosome 13 and contains 12 exons. The RNA junction indicates a fusion between exon 1 of CDKN2D and exon 3 of WDFY2. To identify the genomic breakpoint, a forward primer F1 was designed in exon 1 of CDKN2D and several reverse primers were designed in the intron between exon 2 and 3 of WDFY2. (B) Results from long range PCR on genomic DNA from patient S5 are shown using primer F1 paired with different reverse primers. A product is seen when R2 is used as the reverse primer. (C) Schematic of CDKN2D-WDFY2 genomic breakpoint with the junction sequence and trace identified by Sanger sequencing of the product in (B).
Figure 3
Figure 3. CDKN2D-WDFY2 fusion transcript gives rise to a short WDFY2 protein isoform.
(A) Protein domain structure of CDKN2D and WDFY2. CDKN2D consists of five Ankyrin repeats (AR1-5). WDFY2 contains seven WD-repeats (WD1-7) that are involved in protein-protein interactions and a FYVE domain for binding to phosphatidylinositol-3-phosphate on vesicular membranes. Potential translational consequences of CDKN2D-WDFY2 fusion transcript (plasmid 1) include a truncated CDKN2D protein (7 kD) that starts from the original ATG in CDKN2D ORF (orange) and is fused to an out-of frame exon of WDFY2 (purple), and a short WDFY2 protein (36 kD) that is translated in the original frame starting from an internal cryptic ATG in exon 3 of WDFY2. Plasmid 2 which contains only the ORF for the truncated CDKN2D protein is used as a control. (B) Protein assay of untransfected HEK-293T cells (lane 1 and 4), plasmid 1 transfected (lane 2 and 6), and plasmid 2 transfected cells (lane 3 and 5) with the indicated antibodies. Plasmid 1 transfection led to a 36 kDa protein indicating that short WDFY2 protein isoform is translated (lane 2), while the predicted truncated CDKN2D fusion (7 kD) is not selected for translation (lane 6). Truncated CDKN2D fusion protein is detected only when the expression of plasmid 2 was visualized by anti-FLAG (lane 3) or a commercial anti-CDKN2D antibody (lane 5). Endogenous CDKN2D has a size of 19 kD. Bands at 25 kD and above in lanes 4 to 6 are non-specific bands.
Figure 4
Figure 4. Proteins that are altered consistently in patient tissues and transfected cell lines by short WDFY2.
Heatmaps show the results of a set of 17 proteins identified by RPPA analysis from a total of 130 proteins. These proteins are significantly altered when comparing patient tissue S5 (expressing CDKN2D-WDFY2 fusion transcript) to S19 (does not express fusion transcript), as well as OVCAR8 cell line transfected with short WDFY2 to that transfected with wildtype WDFY2. The antibodies used in this analysis are indicated on the right. Green and red indicate lower and higher expression on a relative scale. The experiment was performed in triplicates. Arrows point to members of PI3K/AKT pathway which are highly represented in this set of 17 proteins (hypergeometric test p-value = 0.0205).

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