Inhibition of Lysyl Oxidase and Lysyl Oxidase-Like Enzymes Has Tumour-Promoting and Tumour-Suppressing Roles in Experimental Prostate Cancer

Abstract

Lysyl oxidase (LOX) and LOX-like (LOXL) enzymes are key players in extracellular matrix deposition and maturation. LOX promote tumour progression and metastasis, but it may also have tumour-inhibitory effects. Here we show that orthotopic implantation of rat prostate AT-1 tumour cells increased LOX and LOXLs mRNA expressions in the tumour and in the surrounding non-malignant prostate tissue. Inhibition of LOX enzymes, using Beta-aminopropionitrile (BAPN), initiated before implantation of AT-1 cells, reduced tumour growth. Conversely, treatment that was started after the tumours were established resulted in unaffected or increased tumour growth. Moreover, treatment with BAPN did not suppress the formation of spontaneous lymph node metastases, or lung tumour burden, when tumour cells were injected intravenously. A temporal decrease in collagen fibre content, which is a target for LOX, was observed in tumours and in the tumour-adjacent prostate tissue. This may explain why early BAPN treatment is more effective in inhibiting tumour growth compared to treatment initiated later. Our data suggest that the enzymatic function of the LOX family is context-dependent, with both tumour-suppressing and tumour-promoting properties in prostate cancer. Further investigations are needed to understand the circumstances under which LOX inhibition may be used as a therapeutic target for cancer patients.

Figure 1. Expression of Lox and Loxl1—Loxl4 mRNAs in orthotopic rat prostate tumours and in the surrounding normal rat prostate tissue.

(a) Relative mRNA expression of Lox and Loxl1—Loxl4 in non-malignant tumour-adjacent rat prostate tissue (n = 8) expressed in relation to the levels of each factor in tumour-free control rat prostate tissue (n = 7) (b) Relative mRNA expression of Lox and Loxl1-Loxl4 in orthotopic AT-1 tumours (n = 8) on day 10 after tumour implantation, expressed in relation to the levels of each factor in the AT-1 tumour cells in vitro. (c) Representative sections from a 10-day orthotopic AT-1 rat prostate tumour (T), tumour-adjacent normal rat prostate tissue (N), and tumour-free control rat prostate tissue stained for LOX (inserts show LOX staining in higher magnifications). (d,e) Relative mRNA expression of Lox and Loxl1-Loxl4 in rat prostate AT-1 tumour cells (d) and in primary rat prostate fibroblasts grown in vitro (e) under normoxic or hypoxic conditions for 24 h. Values are mean ± SEM; *p < 0.05, **p < 0.01.

Figure 2. BAPN treatment of rat prostate tumours.

Rats were injected orthotopically with AT-1 prostate tumour cells (2 × 10³ cells) and treated with an inhibitor of LOX-family enzymes (BAPN, 100 mg/kg) or vehicle (PBS) and tumour weight was analyzed in three different experiments: (a) treatment was started one day before tumour cell implantation and continued on a daily basis until sacrifice at day 10; (b) treatment was started on day 6 when tumours were established, and continued on a daily basis until sacrifice at day 10; and (c) treatment started on day 6 and continued on a daily basis until sacrifice at day 16. Values are mean tumour area (mm²) or mean tumour weight (mg) at sacrifice ± SEM; n = 6–8 per group; *p < 0.05, **p < 0.01. (d) Representative haematoxylin- and eosin-stained sections showing tumour growth in the lymph nodes of both PBS- and BAPN-treated animals (arrowheads indicate the border between lymph node tissue and tumour tissue). (e) Tumour area of spontaneous lymph node metastases in animals with orthotopic tumours treated with BAPN or PBS from day 6 to day 16. Values are mean ± SEM; n = 5 per group; ns: not significant (f) Representative haematoxylin- and eosin-stained sections showing tumour growth in the lungs of both PBS- and BAPN-treated animals. (g) Lung tumour burden in animals injected intravenously with AT-1 cells and treated with a LOX inhibitor (BAPN, 100 mg/kg) or vehicle (PBS) from day 6 to day 16. Values are mean ± SEM; n = 7 per group.

Figure 3. Cell viability of AT-1 tumour cells treated with BAPN or recombinant LOX in vitro.

Cell viability was measured at different time-points using an MTT assay of cultured AT-1 cells incubated with different concentrations of (a) BAPN in normoxia, (b) BAPN in hypoxia, (c) recombinant LOX protein in normoxia, and (d) recombinant LOX in hypoxia. No significant changes were found.

Figure 4. Collagen fibre density in AT-1 tumours and in tumour-adjacent non-malignant prostate tissue.

(a) Representative sections of AT-1 tumours at day 3 and day 10 stained with Sirius red in both regular light microscopy and in polarized light microscopy (Original magnifications 400x, T; tumour). (b) Representative sections of tumour-adjacent prostate tissue at day 7 and 10, contralateral prostate tissue at day 10 and tumour-free control prostate tissue stained with Sirius red and shown in polarized light. (c) Quantification of Sirius red staining in tumour-adjacent prostate tissue and controls. Values are the mean collagen percentage ± SEM; n = 5–7 per group; *p < 0.05, **p < 0.01. (d) Relative mRNA expression of Lox and in AT-1 tumours and in non-malignant tumour-adjacent rat prostate tissue at 3, 6 and 10 days after tumour cell implantation (n = 2–8 per group) expressed in relation to the levels in tumour-adjacent prostate tissue at day 3.