Robo 4 - the double-edged sword in prostate cancer: impact on cancer cell aggressiveness and tumor vasculature

Abstract

Background: The magic roundabout receptor 4 (Robo 4) is a tumor endothelial marker expressed in the vascular network of various tumor entities. However, the role of Robo 4 in prostate cancer (PCa), the second common cause of cancer death among men in -developed countries, has not been described yet. Thus, the present study investigates for the first time the impact of Robo 4 in PCa both in the clinical setting and in vitro. Methods and Results: Immunohistochemical analyses of benign and malignant prostate tissue samples of 95 PCa patients, who underwent radical prostatectomy (RPE), revealed a significant elevated expression of Robo 4 as well as its ligand Slit 2 protein in cancerous tissue compared to benign. Moreover, increased Robo 4 expression was associated with higher Gleason score and pT stage. In advanced stage we observed a hypothesis-generating trend that high Robo 4 and Slit 2 expression is associated with delayed development of tumor recurrence compared to patients with low Robo 4 and Slit 2 expression, respectively. In contrast to so far described exclusive expression of Robo 4 in the tumor vascular network, our analyses showed that in PCa Robo 4 is not only expressed in the tumor stroma but also in cancer epithelial cells. This finding was also confirmed in vitro as PC3 PCa cells express Robo 4 on mRNA as well as protein level. Overexpression of Robo 4 in PC3 as well as in Robo 4 negative DU145 and LNCaP PCa cells was associated with a significant decrease in cell-proliferation and cell-viability. Conclusion: In summary we observed that Robo 4 plays a considerable role in PCa development as it is expressed in cancer epithelial cells as well as in the surrounding tumor stroma. Moreover, higher histological tumor grade was associated with increased Robo 4 expression; controversially patients with high Robo 4 tend to exert lower biochemical recurrence possibly reflecting a protective role of Robo 4.

Keywords: Prostate cancer; Robo 4; Slit 2; cancer aggressiveness; tumor recurrence.

Figure 1

Immunohistochemical analyses of A) Robo 4-, B) Slit 2-staining intensity scores as well as C) CD31 microvessel density (MVD) of radical prostatectomy specimens analyzed according to benign vs. cancer tissue. Robo 4 histology score comparing D) Gleason score (GS), E) GS upgrading and F) pathological stage in the radical prostatectomy specimens. *p<0.05; **p<0.01; ***p<0.001; n=95.

Figure 2

Robo 4 staining intensity scores of a tissue microarray of radical prostatectomy specimens of prostate cancer patients stratified according to cell compartments: A) stroma and tumor cell in cancer cores; B) Robo 4 expression in the stromal compartment of benign and cancer cores; ***p<0.001; n=95.

Figure 3

Representative pictures of Hematoxilin/Eosin (HE) and immunhistochemical stainings (AMACR/p63 doublestaining, CD31, Robo 4 on paraffin embedded tissue of a radical prostatectomy specimen. A) HE staining with clearly different morphology in benign and cancerous glands. B) P63/AMACR doublestaining demonstrating benign glands as AMACR negative with p63 positive basal cells (dark brown), while cancer glands are AMACR positive (red) and p63 negative. C) Endothelial cells with typical CD31 positivity. D) Robo 4 expression is missing/weak in benign and intermediate/strong in cancer. In addition, endothelial and stromal cells show Robo 4 positivity (in cancer glands surrounding stroma stronger than in benign glands surrounding stroma). Scale bar = 200 μm.

Figure 4

Box plots showing A) Robo 4 and B) Slit 2 expression in patients with a biochemical recurrence (BCR) after radical prostatectomy. Data represent mean + SEM. Kaplan Meier curves of high (green) versus low (blue) C) Robo 4 and D) Slit 2 expression. Differences among both groups were applied by log-rank test. 75% quartile was used for determination of “high” Robo 4 or Slit 2 expression; n=16.

Figure 5

Kaplan Meier curves analyzing overall survival of patients with high (green) versus low (blue) A) Robo 4 and B) Slit 2 expression validated in an external dataset (TCGA_PRAD, race white); n=147.

Figure 6

A) Robo 4 mRNA expression in different prostate cancer cell lines as well as in HUVEC used as control cell line, n=3; B) Robo4 protein expression on fixed embedded PCa PC3 and LNCaP cells.

Figure 7

A) % Viability and B) % Cell proliferation upon overexpression of Robo 4 in PC3, DU145 and LNCaP cells; data from ≥3 independent experiments, *p<0.05; **p<0.01; ***p<0.001.