The increased expression of fatty acid-binding protein 9 in prostate cancer and its prognostic significance

Abstract

In contrast to numerous studies conducted to investigate the crucial role of fatty acid binding protein 5 (FABP5) in prostate cancer, investigations on the possible involvement of other FABPs are rare. Here we first measured the mRNA levels of 10 FABPs in benign and malignant prostate cell lines and identified the differentially expressed FABP6 and FABP9 mRNAs whose levels in all malignant cell lines were higher than those in the benign cells. Thereafter we assessed the expression status of FABP6 and FABP9 in both prostate cell lines and in human tissues. FABP6 protein was overexpressed only in 1 of the 5 malignant cell lines and its immunostaining intensities were not significantly different between benign and malignant prostate tissues. In contrast, FABP9 protein was highly expressed in highly malignant cell lines PC-3 and PC3-M, but its level in the benign PNT-2 and other malignant cell lines was not detectable. When analysed in an archival set of human prostate tissues, immunohistochemical staining intensity for FABP9 was significantly higher in carcinomas than in benign cases and the increase in FABP9 was significantly correlated with reduced patient survival times. Moreover, the increased level of staining for FABP9 was significantly associated with the increased joint Gleason scores (GS) and androgen receptor index (AR). Suppression of FABP9 expression in highly malignant PC3-M cells inhibited their invasive potential. Our results suggest that FABP9 is a valuable prognostic marker to predict the outcomes of prostate cancer patients, perhaps by playing an important role in prostate cancer cell invasion.

Keywords: FABP9; PSA; gleason scores; prognosis; prostate cancer.

Figure 1. Quantitative PCR analysis of levels of FABP mRNAs in benign and malignant prostate epithelial cells

Benign cell line PNT-2, weakly malignant cell line LNCaP, moderately malignant cell line 22RV-1 and highly malignant cell lines Du145, PC-3 and PC3-M were cultured to 80% confluence and harvested for mRNA extraction. The quality of the mRNA was assessed by a 2100 Bio-analyser. A. the electrophernograms of the gel-like images of mRNAs from 6 cell lines and from the RNA ladder (marker). B. the double peaks representing 18S and 28S sub-units and the RNA integrity numbers (RIN) of the samples from different cell lines. C. relative levels of FABP mRNAs detected in benign and malignant prostate cell lines.

Figure 2. Expression of FABP6 and FABP9 in prostate cell lines and tissues

A. Measurement of FABPs in benign and malignant prostate cells. a. Western blot analysis of FABP6 in benign and malignant prostate cells. b. relative levels of FABP6 in different cell lines. The level of FABP6 in the benign PNT-2 cells was set at 1.0; levels in other cell lines were obtained by relating to that in PNT-2 cells. The positive control (MCF-7) was suggested by the manufacturer of the antibody. The results were obtained from 3 separate experiments (mean ± SD). c. Western blot analysis of FABP9 in benign and malignant prostate cells. d. relative levels of FABP9 in different cell lines. Since the expression in PNT-2, LNCaP, 22RV-1 and Du145 is not detectable, their levels were expressed as “0”. The level in PC-3 cells was set at 1.0, the level in PC3-M cells was obtained by relating to that in PC-3. The results were obtained from 3 separate experiments (mean ± SD). B. Immunohistochemical detection of FABP6 and FABP9 in benign and malignant prostate tissues. Positive controls for FABP6 and FABP9 were breast cancer tissue and normal kidney tissue respectively (x100). For anti-FABP6, only nuclear staining was observed (identified in the insert by the arrow). For anti-FABP9, only cytoplasmic staining was observed (identified in the insert by the arrow). Carcinomas were divided into three categories according to their combined GS: weakly malignant (or GS ≤5), moderately (or GS 6-7) and high malignant (or GS 8-10) tissues.

Figure 3. Kaplan-Meier survival curves of patients with prostate cancer

The cumulative survival of patients was plotted against the time in months for different levels of 4 parameters. A. different GS: group 1, GS ≤5 (n=17); group 2, GS 6 to 7 (n=38); group 3, GS 8 to 10 (n=45). B. different AR indices: low group, <4 (n=41); moderate group, 4-6 (n=50); high group, 6-9 (n=11). C. different levels of PSA: group 1, PSA <10 ng/ml (n=38) and group 2, PSA ≥10 ng/ml (n=64). D. different levels of staining for FABP9: group 1, weakly positive (n=28); group 2, moderately positive (n=38); group 3, strongly positive (n=21).

Figure 4. Box plot of correlations amongst 3 different variables

A. Correlation between the stain intensity for FABP9 and the Gleason score (GS) in 3 groups of patients with prostate cancer: GS < 6 (low), GS 6 to 7 (moderate) and GS 8 to 10 (high). B. Correlation between stain intensity for FABP9 and androgen receptor (AR) index in 3 groups of patients with prostate cancer: weak (W), moderate (M) and high (S) levels of FABP9. C. Correlation between stain intensity for FABP9 and prostatic specific antigen (PSA) level in 3 groups of patients with prostate cancer: weak (W), moderate (M) and high (S) levels of FABP9.

Figure 5. Knockdown of FABP9 mRNA in highly malignant prostate cancer cells and testing the invasiveness of the transfectants expressing reduced levels of FABP9

A. Effect of mRNA knockdown on FABP9 expression in PC3-M cells. a. Western blot analysis of FABP9 expressed in PC3-M cells and cells transiently transfected with 3 different siRNAs. To standardize the immune reactions, β-actin antibody was incubated with the same blot. b. relative levels of FABP9 in PC3-M cells after transient transfection. The level of FABP9 in parental PC3-M was set at 1.0 and levels expressed in other transiently-transfected cells with the controls and different siRNAs were obtained by relating to that in parental cells. c. Western blot analysis of FABP9 in control PC3-M cells and 5 different clones generated by transfection with the shRNA based on siRNA-3. To standardize the immune reactions, β-actin antibody was incubated with the same blot. d. The relative levels of FABP9 in PC3-M transfected with scramble shRNA control was set at 1.0 and levels expressed in other transfected cell lines were obtained by comparing with the control. e. Western blot analysis of FABP5 in transfectants expressing reduced levels of FABP9. f. The relative levels of FABP5 in PC3-M cells and transfectants expressing reduced levels of FABP9. B. The effect of FABP9 suppression on invasiveness of transfectant cells. The results (mean ± SD) were obtained from 3 separate experiments. Insert picture: 3 panels represent the invasiveness of the control, moderately suppressed (M) and highly suppressed (H) cells respectively.