Aldehyde dehydrogenase 3A1 associates with prostate tumorigenesis

Abstract

Background: Accumulating evidence demonstrates high levels of aldehyde dehydrogense (ALDH) activity in human cancer types, in part, because of its association with cancer stem cells. Whereas ALDH1A1 and ALDH7A1 isoforms were reported to associate with prostate tumorigenesis, whether other ALDH isoforms are associated with prostate cancer (PC) remains unclear.

Methods: ALDH3A1 expression was analysed in various PC cell lines. Xenograft tumours and 54 primary and metastatic PC tumours were stained using immunohistochemistry for ALDH3A1 expression.

Results: In comparison with the non-stem counterparts, a robust upregulation of ALDH3A1 was observed in DU145-derived PC stem cells (PCSCs). As DU145 PCSCs produced xenograft tumours with more advanced features compared with those derived from DU145 cells, higher levels of ALDH3A1 were detected in the former; a dramatic elevation of ALDH3A1 occurred in DU145 cell-derived lung metastasis compared with local xenograft tumours. Furthermore, while ALDH3A1 was not observed in prostate glands, ALDH3A1 was clearly present in PIN, and further increased in carcinomas. In comparison with the paired local carcinomas, ALDH3A1 was upregulated in lymph node metastatic tumours; the presence of ALDH3A1 in bone metastatic PC was also demonstrated.

Conclusions: We report here the association of ALDH3A1 with PC progression.

Figure 1

Expression of ALDH1A1 and ALDH3A1 in prostate epithelial and carcinoma cells. (A) Cell lysates and RNA were collected and isolated from the indicated cell lines and examined with western blot analysis for protein expression of ALDH1A1 (top) and with real-time PCR for mRNA levels (bottom). (B) Expression of ALDH3A1 was also examined for protein (top) and mRNA expression (bottom). β-actin was used as an internal control. Relative transcript abundance was determined according to a published procedure (van den Hoogen et al, 2010).

Figure 2

Upregulation of ALDH3A1 in DU145 prostate cancer stem cells. Prostate cancer-enriched stem-like cells were isolated as spheres from DU145. (A) Cell lysates were collected and western blot analysis was carried out for DU145 and DU145 spheres to determine ALDH3A1 expression (top). RNA was isolated from both cell lines and examined for ALDH3A1 mRNA expression using real-time PCR (bottom). β-actin was used as an internal control. ALDH3A1 mRNA in DU145 spheres is shown as a fold change to DU145 cells (mean±s.d.). *P<0.05 by a Mann–Whitney test. (B) DU145 spheres were embedded in paraffin, sectioned and stained with immunohistochemistry for ALDH3A1 and ALDH1A1. Small intestine was used as a positive control.

Figure 3

Specific increase in ALDH3A1 expression compared with DU145 in prostate cancer stem cells. (A) RNA was isolated from DU145 prostate cancer stem cells and DU145 cells and a microarray were carried out by the University Health Network Microarray Facility. Fold change was calculated as a ratio of the signal intensity of DU145 prostate cancer stem cells to DU145. A fold change of 1 (red line) denotes that both cell lines have relatively the same signal intensity. (B) Real-time PCR was carried out on all 19 ALDH genes for DU145 and DU145 prostate cancer stem cells. β-actin was used as an internal control. Corresponding ALDH mRNA in DU145 prostate cancer stem cells are shown as a fold change to DU145 cells (mean±s.d.).

Figure 4

Expression of ALDH1A1 and ALDH3A1 in prostate cancer xenograft tumours. Xenograft tumours derived from DU145 (n=3) and DU145 spheres (n=3) through subcutaneous injections into immunocompromised mice were stained for ALDH1A1 and ALDH3A1 using immunohistochemistry. Human small intestines were used as a positive control for ALDH1A1.

Figure 5

Expression of ALDH1A1 and ALDH3A1 in metastatic prostate cancer xenograft tumours. (A) A total of 0.3 ml of 10⁶ DU145 cells in PBS was injected intravenously through the tail vein of immunocompromised mice. Lungs were harvested at 16 weeks post injection and tumours can be seen in lungs injected with DU145 cells (black arrows). (B) Lung tissues with metastatic nodules were embedded in paraffin, sectioned and stained for ALDH1A1 and ALDH3A1 (n=2).

Figure 6

ALDH3A1 but not ALDH1A1 is expressed in primary prostate cancer tissues. (A) Primary prostate cancer tissues were stained for ALDH1A1 (n=6) and (B) ALDH3A1 (n=47). Typical images are shown for normal, PIN, Gleason 6 and 9 carcinomas.

Figure 7

ALDH3A1 is associated with prostate cancer progression. (A) A total of 47 primary prostate cancer tissues were IHC-stained for ALDH3A1 (normal n=6, PIN n=6, Gleason 5–7 n=24, Gleason 8–10 n=23). The H-score was calculated for each patient and grouped into normal, PIN or carcinoma. Staining intensity according to the H-score was graphed (mean±s.d.). Note: the H-score for normal was close to 0. (B) Percentage of patients examined expressing negative to weak or positive ALDH3A1 staining was calculated. (C) Primary prostate cancer tissue was stained for ALDH3Al. Regions negative for staining in normal glands, weak staining in PIN and strong staining in carcinoma can be observed. (D) The percentage of patients expressing negative to weak or positive staining with low Gleason or high Gleason prostate cancer was examined and calculated.

Figure 8

ALDH3A1 is expressed in metastatic prostate cancer. (A) Three primary prostate cancer patients with lymph node metastasis were examined for ALDH1A1. (B) Expression for ALDH3A1 was also examined in the three primary prostate cancer tissue and their corresponding lymph node metastatic prostate cancer. (C) Four prostate cancer bone metastatic tumours were stained and examined for ALDH3A1 expression. Representative images for two patient samples are shown.