Olfactomedin 4 Suppresses Prostate Cancer Cell Growth and Metastasis via Negativ

人嗅球蛋白4基因(OLFM4)编码一种与嗅球蛋白相关的糖蛋白。Olfm4通常在包括前列腺在内的有限数量的组织中表达，但它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，我们发现在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基因转染PC-3前列腺癌细胞，建立了表达OLFM4的PC-3细胞克隆。表达OLFM4的PC-3细胞克隆的体外增殖和侵袭力较载体转染组PC-3细胞明显降低。此外，裸鼠注射了表达OLFM4的PC-3细胞，与注射了载体的对照细胞相比，肿瘤生长和骨侵袭转移都有所减少。机制研究表明，OLFM4可能通过靶向组织蛋白酶D调节细胞自噬而发挥抗癌作用，因为OLFM4降低了组织蛋白酶D的蛋白水平和酶活性，并减弱了组织蛋白酶D诱导的癌细胞增殖。此外，在Matrigel侵袭实验中，过表达OLFM4可抑制基质细胞衍生因子-1(SDF-1)诱导的PC-3细胞侵袭，部分是通过阻断SDF-1介导的AKT磷酸化。在表达OLFM4的PC-3细胞中的免疫共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负向相互作用，抑制前列腺癌生长和骨转移。 为了进一步研究OLFM4基因在前列腺组织中的生理功能，我们建立并分析了OLFM4基因敲除的小鼠模型。在过去的一年里，我们发现在临床样本和小鼠模型中，OLFM4缺失与前列腺癌的进展有关。在患者样本中，我们用激光捕获显微切割技术发现癌细胞中olfm4基因外显子2内的rs2298231(S118S)杂合性缺失。通过荧光原位杂交分析，我们进一步在前列腺癌样本中发现了OLFM4基因的半合子和纯合子缺失。有趣的是，OLFM4基因敲除小鼠在1824个月大时患上了前列腺上皮内瘤变(44%)和前列腺癌(28%)，表达芯片分析表明这些前列腺样本中与细胞增殖相关的基因水平增加。特别是，OLFM4的缺失导致ETS家族基因Ets1和促癌基质金属蛋白酶19(MMP19)的表达增加。综上所述，这些数据表明，OLFM4基因的缺失促进了前列腺上皮细胞的肿瘤进展。

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.