Pseudogene Associated Recurrent Gene Fusion in Prostate Cancer

Abstract

We present the functional characterization of a pseudogene associated recurrent gene fusion in prostate cancer. The fusion gene KLK4-KLKP1 is formed by the fusion of the protein coding gene KLK4 with the noncoding pseudogene KLKP1. Screening of a cohort of 659 patients (380 Caucasian American; 250 African American, and 29 patients from other races) revealed that the KLK4-KLKP1 is expressed in about 32% of prostate cancer patients. Correlative analysis with other ETS gene fusions and SPINK1 revealed a concomitant expression pattern of KLK4-KLKP1 with ERG and a mutually exclusive expression pattern with SPINK1, ETV1, ETV4, and ETV5. Development of an antibody specific to KLK4-KLKP1 fusion protein confirmed the expression of the full-length KLK4-KLKP1 protein in prostate tissues. The in vitro and in vivo functional assays to study the oncogenic properties of KLK4-KLKP1 confirmed its role in cell proliferation, cell invasion, intravasation, and tumor formation. Presence of strong ERG and AR binding sites located at the fusion junction in KLK4-KLKP1 suggests that the fusion gene is regulated by ERG and AR. Correlative analysis of clinical data showed an association of KLK4-KLKP1 with lower preoperative PSA values and in young men (<50 years) with prostate cancer. Screening of patient urine samples showed that KLK4-KLKP1 can be detected noninvasively in urine. Taken together, we present KLK4-KLKP1 as a class of pseudogene associated fusion transcript in cancer with potential applications as a biomarker for routine screening of prostate cancer.

Figure 1

The structure of KLK4-KLKP1 fusion and the RNA-ISH screening of KLK4-KLKP1 in tissue micro arrays. (A) Schematic diagram showing the structure of KLK4-KLKP1 fusion. KLK4-KLKP1 is formed through the fusion of exon 1 and 2 of KLK4 gene with exon 4 and 5 of KLKP1. (B) The predicted sequence of KLK4-KLKP1 fusion protein. The sequence in purple is derived from KLK4, while the sequence in red is originating from KLKP1. (C) The expression of KLK4-KLKP1 in prostate tissue cores detected by RNA-ISH. The bottom set of images shows an enlarged section of the corresponding tissue core in the top set of images. Values 1+ to 4+ indicate the intensity of KLK4-KLKP1 RNA-ISH staining. (D) Prostate cancer specific expression of KLK4-KLKP1. KLK4-KLKP1 RNA-ISH staining in benign, HGPIN, and prostate cancer tumor cores is shown. The bottom set of images contains a magnified area of the images on the top. Values 1+ to 4+ refer to the intensity of the KLK4-KLKP1 RNA-ISH staining. (E) KLK4-KLKP1 is expressed more in the prostate cancer patients (Gleason Grade Group 1-5) compared to noncancer (benign, HGPIN, atypical, and stroma) cases. The percentage of cases showing a positive KLK4-KLKP1 RNA-ISH signal among noncancer and Gleason Grade Group1-5 is shown. P value was calculated based on Pearson's chi-square test. (F) KLK4-KLKP1 is expressed more in young prostate cancer patients. The percentages of cases with positive KLK4-KLKP1 RNA-ISH signal in the young patient (age lower than 50 years) and old patient groups (age equal to or higher than 50 years) are shown. P value was calculated based on Pearson's chi-square test. (G) KLK4-KLKP1 expression is associated with ERG overexpression. SPINK1, ETV1, ETV4, and ETV5 overexpression is mutual from KLK4-KLKP1 expression. PTEN loss is significantly lower in cases with KLK4-KLKP1 expression. The percentages of cases showing positive signal for ERG, SPINK1, ETV1, ETV4, ETV5, or PTEN loss among KLK4-KLKP1 RNA-ISH positive cases (dark gray bars) and KLK4-KLKP1 RNA-ISH negative cases (light gray bars) are shown. P value was calculated based on Pearson's chi-square test. Abbreviations: GG, Gleason Grade Group; HGPIN, high-grade prostate intraepithelial neoplasia.

Figure 2

Validation of the expression of KLK4-KLKP1 protein in HEK-293 cells and PDX tissues. (A) The qRT-PCR analysis HEK-293 cells transfected with and without FLAG tagged-KLK4-KLKP1. HEK-293 cells were transfected with adenoviral vectors carrying FLAG tagged-KLK4-KLKP1 (adeno-FLAG-KLK4-KLKP1). As a control, untransfected cells treated with bortezomib were used. The expression of KLK4-KLKP1 was confirmed by qRT-PCR. (B) Western blot analysis of HEK-293 cells transfected with FLAG-tagged KLK4-KLKP1 using anti-FLAG, anti–KLK4-KLKP1, and anti–β-actin antibody. (C) Full images of Western blot analysis of Figure 2B. The full anti-Flag blot (left side) and the full anti–KLK4-KLKP1 blot (right side) are shown. The molecular weight ladder is also shown on the left. The arrowhead indicates KLK4-KLKP1 protein observed at the expected molecular weight. (D) Western blot analysis of KLK4-KLKP1 qRT-PCR negative (MDA PCa144-13) and qRT-PCR positive (MDA PCa 153-7) PDX tissues using anti–KLK4-KLKP1 and anti–β-actin antibody. (E) Full image of Western blot analysis of Figure 2D. The arrowhead shows a band observed around the expected molecular weight only in the KLK4-KLKP1 qRT-PCR positive tissue sample (MDA PCa 153-7). (F) IHC staining of KLK4-KLKP1 qRT-PCR positive and qRT-PCR negative PDX models. (G) Representative images from a TMA stained with KLK4-KLKP1 RNA ISH probe (left) and KLK4-KLKP1 antibody (right). Comparison between IHC staining with anti-KLK4-KLKP1 antibody and RNA-ISH on two representative TMA tissue cores. The IHC staining with anti–KLK4-KLKP1 on a KLK4-KLKP1 RNA ISH positive tumor core (left side) and a KLK4-KLKP1 RNA ISH negative tumor core is shown. The inner circles show the enlarged images of a tumor area of the TMA tissue core.

Figure 3

Functional characterization of KLK4-KLKP1. (A) qRT-PCR validation of KLK4-KLKP1 expression in RWPE-1 cells after stable transfection with FLAG tagged-KLK4-KLKP1. As controls, untransfected cells (control) and cells transfected with LacZ were used. (B) Analysis of cellular proliferation in RWPE-1 cells stably expressing FLAG tagged KLK4-KLKP1. Cells were plated in 96-well plates. The number of cells was measured on days 2, 4, 6, and 8 using a Coulter particle counter. Cells untransfected and transfected with LACZ were used as controls. (C) Analysis of cell invasion in RWPE-1 cells. The invasion of RWPE-1 cells stably transfected with either FLAG tagged-KLK4-KLKP1 or LacZ was studied using the Boyden chamber assay. Untransfected cells were also used as a control. After invasion of cells into the invasion chamber, cells were fixed and visualized using crystal violet. Additionally, the invasion chamber membranes carrying the fixed cells were dipped in glacial acetic acid, and the absorbance at 560 nm was also measured. Representative images of the crystal violet–stained cells that underwent invasion in each case and the absorbance at 560 nm are shown. (D) Analysis of cell invasion in PrEC cells. The cellular invasion in PrEC cells transfected with FLAG tagged-KLK4-KLKP1 was performed as described in panel C. The number of invaded cells was counted and plotted. In addition to LACZ and untransfected cells, PrEC cells transfected with EZH2 were also used a control. (E) Intravasation of RWPE-1 cells measured using CAM assay. RWPE-1 cells stably transfected with FLAG tagged-KLK4-KLKP1 were implanted on eggs. The presence of intravasated cells in the lower CAM was assessed by quantitative human Alu-specific PCR. Untransfected cells and cells transfected with LACZ were used as controls. (F) Analysis of weight of extraembryonic tumors isolated from eggs implanted with RWPE-1 cells stably expressing FLAG-tagged KLK4-KLKP1. Cells transfected with LACZ and untransfected cells were used as controls. Abbreviations: CAM, chicken chorioallantoic membrane.

Figure 4

Gene expression analysis of KLK4-KLKP1. (A) Heat map showing the top 100 genes differentially expressed in RWPE-1 cells stably transfected with KLK4-KLKP1 compared to cells transfected with LACZ. The results from two independent trials are shown. (B) Gene set enrichment analysis of differentially expressed genes. The genes were enriched in two curated gene sets, one involving genes upregulated in endometroid endometrial metastatic tumor “BIDUS_METASTASIS_UP” (top image) and the other including genes overexpressed in melanoma metastatic cancer “WINNEPENNINCKX_METASTASIS_UP” (bottom image). (C) Top 10 KEGG pathways enriched in differentially expressed genes obtained using DAVID tool.