Olfactomedin 4 Suppresses Prostate Cancer Cell Growth and Metastasis via Negativ

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The human olfactomedin 4 gene (OLFM4) encodes an olfactomedin-related glycoprotein. OLFM4 is normally expressed in a limited number of tissues, including the prostate, but its biological functions in prostate are largely unknown. In this study, we found that OLFM4 messenger RNA was reduced or undetectable in prostate cancer tissues and prostate cancer cell lines. To study the effects of OLFM4 on prostate cancer progression, we transfected PC-3 prostate cancer cells with OLFM4 to establish OLFM4-expressing PC-3 cell clones. The OLFM4-expressing PC-3 cell clones were found to have decreased proliferation and invasiveness compared with vector-transfected control PC-3 cells in vitro. In addition, nude mice injected with OLFM4-expressing PC-3 cells demonstrated reduced tumor growth and bone invasion and metastasis compared with mice injected with vector-transfected control cells. Mechanistic studies revealed that OLFM4 may exhibit its anticancer effects through regulating cell autophagy by targeting cathepsin D, as OLFM4 reduced cathepsin D protein levels and enzymatic activity and attenuated cathepsin D-induced cancer cell proliferation. In addition, overexpression of OLFM4 abrogated stromal cell derived factor-1 (SDF-1)-induced PC-3 cell invasiveness in a Matrigel invasion assay, partially through blocking SDF-1-mediated AKT phosphorylation. Coimmunoprecipitation and immunofluorescence staining studies in OLFM4-expressing PC-3 cells demonstrated a direct interaction between OLFM4 and cathepsin D or SDF-1. Taken together, these results suggest that OLFM4 negatively interacts with cathepsin D and SDF-1 and inhibits prostate cancer growth and bone metastasis. To further investigate the physiological functions of OLFM4 gene in the prostate tissue, we have developed and analyzed an OLFM4 knock out mouse model. We have observed that homozygous OLFM4 mutant mice develop prostatic epithelial hyperplasia in 3-6 months, prostatic intraepithelial neoplasia (PIN) in 10 to 12 months and tumor in 18-20 months mice anterior (AP) and dorsal-lateral prostate (DLP). A comparable number of OLFM4 +/+ were studied, none of whom developed tumors, and the incidence of PIN was reduced by 60% at similar time points. Immunohistochemical staining showed that the tumors from OLFM4 mutant (-/-) mice expressed androgen receptor but not synaptophysin which is neuroendocrine cells marker or 34E12 and p63 which are basal cell markers. Consisent with the aggressive nature of these tumors, a high proportion of the cells were Ki67 positive and revealed abnormal E-cadherin expression. The invasive tumor in the anterior prostate (AP) of 20 month old OLFM4 null mouse were easily apparent with HE and SMA staining. Locally invasive tissues in the dorsal lateral prostate (DLP) of 20 month OLFM4 null mouse and lung metastasized tumor tissues were identified and positive for androgen receptor, Ki-67 and E-cadherin immunohistochemical staining. Ki-67 immunohistochemical staining was used as an indicator of increased proliferation of prostatic epithelial cells. We found that the ratio ofKki-67 positive cells was significantly higher in the prostate epithelial cells from DLP of 12 and 20 months OLFM4 null mice than control mice. The Ki 67 positive cells were increased in the PIN and tumor. These results suggest that OLFM4 gene knock out leading to increasing of proliferative abilities of mouse prostatic epithelial cells. Preliminary data implicate upregulation of the AKT/GSK3beta/beta-catenin signaling pathways and increased expression of cyclin D1 and c-Myc as early as 3-4 months in the prostate tissue of the OLFM4 null mice.