Our study was the first to demonstrate the anticancer functions of OLFM4 in human prostate cancer. Firstly, we found that OLFM4 was highly expressed in human normal prostate tissues and normal prostate cell lines and was moderately expressed in human benign prostatic hyperplasia tissue samples but was significantly reduced or lost in human prostate cancer tissues and cancer cell lines. Secondly, increased expression of human OLFM4 in PC-3 cells led to tumor growth inhibition in vitro and in xenografts in nude mice, and, most impressively, led to the inhibition of cancer cell bone invasion and multiple organ metastasis in vivo. Thirdly, OLFM4-mediated tumor inhibition was associated with a negative interaction with cathepsin D and SDF-1.
High levels of OLFM4
mRNA expression have been described in several tumor tissues (24
), and a positive correlation between OLFM4
mRNA with stomach cancer progression and disease stage has been observed (25
). However, we found OLFM4
mRNA expression is significantly reduced or lost in human prostate cancer. The published database (Gene Card) also showed a similar expression pattern in human normal prostatic tissues and prostate cancer. These results suggest that the pattern of OLFM4
gene expression is dependent on tumor type. On the other hand, using an OLFM4-specific antibody, we found OLFM4 protein was highly expressed in well-differentiated tumors and was decreased in most undifferentiated colon, stomach (29
) and prostate cancers (data not shown).
More recently, Conrotto et al.
reported that OLFM4 protein was detected in only one of five prostate cancer samples tested using an OLFM4 monoclonal antibody and that fewer than 15% of the tumor cells stained positively in that positive case. They also found that 64% of colorectal tumors expressed OLFM4 protein, but no correlation with tumor cell differentiation was reported (31
). Although a high level of OLFM4
mRNA was detected in several tumor tissues compared with that in normal tissues, the role of the OLFM4
gene in human cancer initiation and progression was not clear. Our previous studies have demonstrated that OLFM4 binds to cell-surface adhesion molecules, such as lectins and cadherin, facilitating cell adhesion in HEK 293 cells (20
). Overexpression of OLFM4
in the HT-29 colon cancer cell lines altered filamentous actin distribution, cell shape and cell adhesion but did not inhibit cancer cell growth (30
). In this study, we showed that OLFM4
-expressing PC-3 cell clones reduced cell growth, invasiveness and metastasis. In contrast, a previous study reported that overexpression of the OLFM4
gene in mouse prostate Tramp-C1 cells promoted tumor growth in syngeneic mice (32
). Although these studies used different technical approaches from our studies that may account for the different results obtained, the differences in results also suggest that OLFM4
may play dual roles in tumor cell growth, depending on gene expression level and cellular context.
Cathepsin D plays a number of important roles underlying physiological and pathological conditions (38
). It has been reported that cathepsin D is involved in regulating cell autophagy, a lysosomal degradation pathway that regulates many physiological and pathological processes, including cancer (39
). In the present study, we demonstrated that OLFM4 protein physiologically interacts with cathepsin D and regulates cathepsin D autocatalytic activity, as well as blocks cathepsin D biological functions. We also found that OLFM4
-expressing PC-3 clones had significantly increased cell autophagy compared with vector-transfected control PC-3 cells. It is possible that OLFM4
may exhibit its inhibitory effects on cancer cell growth through regulating cell autophagy in OLFM4
-expressing PC-3 cells. Although many factors are involved in the regulation of the autophagic pathway, we postulate that cathepsin D is one of the OLFM4
target molecules involved in the regulation of autophagy in OLFM4
-expressing PC-3 cells. Our results suggest that the OLFM4
gene may be involved in the regulation of cell autophagy in human prostate cancer cells.
The participation of SDF-1/CXCR4 in prostate cancer invasion and bone metastasis has recently been documented (40
) and neutralizing anti-CXCR4 antibody has also been reported to block metastasis and growth of PC-3 cancer cells in osseous sites (42
). Moreover, it has been reported that chemokine interactions could modulate SDF-1-mediated prostate cancer progression and metastasis (43
). In the current study, we have identified a direct binding of OLFM4 and SDF-1 and their colocalization in prostate cancer cells. We also found that the interaction between OLFM4 and SDF-1 influenced SDF-1/CXCR4 signaling, resulting in prostate cancer cell growth and bone metastasis. Our results did not completely reveal how SDF-1 interacts with intracellular or extracellular OLFM4 and other unidentified matrix components of SDF-1/CXCR4 signaling during prostate cancer progression. However, our data are sufficient to suggest that, as with its interaction with cathepsin D, OLFM4 was able to attenuate SDF-1/CXCR4 signaling-mediated tumor cell growth and bone invasion. SDF-1 and other growth factors may have multiple binding partners for modulating optimal oncogenic signaling in prostate tissues (47
), but the interaction between SDF-1 and OLFM4 may have unique functions that lead to decreased sequestration of SDF-1 to the cell surface, a reduced chemokine gradient for tumor cell migration, or quaternary structures and oligomerization status of SDF-1 binding of OLFM4 that alter SDF-1 mitogenic activity. It is also possible that cell surface OLFM4 modulates SDF-1 function in vivo
by forming a dimeric complex with the receptor and then inhibiting SDF-1/CXCR4 downstream signal transduction.
Taken together, this study has identified a novel anticancer function of OLFM4 by demonstrating that OLFM4 suppresses human prostate cancer cell growth and metastasis via negative interaction with cathepsin D and SDF-1. Therefore, OLFM4 may have therapeutic potential for prostate cancer therapy and metastasis prevention.