Olfactomedin 4 Suppresses Prostate Cancer Cell Growth and Metastasis via Negativ

人类嗅消素4基因（OLFM4）编码一种与嗅消素相关的糖蛋白。OLFM4通常在包括前列腺在内的有限组织中表达，但其在前列腺中的生物学功能在很大程度上是未知的。在本研究中，我们发现前列腺癌组织和前列腺癌细胞系中OLFM4信使RNA减少或检测不到。为了研究OLFM4对前列腺癌进展的影响，我们用OLFM4转染PC-3前列腺癌细胞，建立表达OLFM4的PC-3细胞克隆。与载体转染的对照PC-3细胞相比，表达olfm4的PC-3细胞克隆的体外增殖和侵袭性均降低。此外，与注射载体转染的对照细胞相比，注射表达olfm4的PC-3细胞的裸鼠肿瘤生长和骨侵袭转移减少。机制研究表明，OLFM4可能通过靶向组织蛋白酶D调控细胞自噬发挥抗癌作用，降低组织蛋白酶D蛋白水平和酶活性，减弱组织蛋白酶D诱导的癌细胞增殖。此外，在Matrigel侵袭实验中，过度表达OLFM4可通过阻断SDF-1介导的AKT磷酸化，部分消除基质细胞衍生因子-1 （SDF-1）诱导的PC-3细胞侵袭性。在表达OLFM4的PC-3细胞中进行的共免疫沉淀和免疫荧光染色研究表明，OLFM4与组织蛋白酶D或SDF-1直接相互作用。综上所述，这些结果表明OLFM4与组织蛋白酶D和SDF-1负相互作用，抑制前列腺癌的生长和骨转移。

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.During this last year, we found that OLFM4 loss is associated with prostate cancer progression in clinical samples and in a mouse model. In patient specimens, we found loss of heterozygosity at rs 2298231(S118S) within exon 2 of OLFM4 in cancerous prostate epithelial cells using laser-capture microdissection. We further found hemizygous and homozygous deletions of the OLFM4 gene in prostate cancer samples via fluorescence in situ hybridization analysis. Interestingly, OLFM4 knockout mice developed prostatic intraepithelial neoplasia (44%) and prostatic adenocarcinoma (28%) at ages 1824 months, and expression microarray analysis demonstrated increased levels of genes associated with cell proliferation in these prostate samples. In particular, loss of OLFM4 produced increased expression of the ETS family gene Ets1 and the cancer-promoting matrix metalloproteinase 19 (MMP19). Taken together, these data indicate that loss of the OLFM4 gene facilitates neoplastic progression in prostatic epithelial cells.