Olfactomedin 4 Suppresses Prostate Cancer Cell Growth and Metastasis via Negativ

人嗅觉调节蛋白4基因（OLFM 4）编码一种嗅觉调节蛋白相关糖蛋白。OLFM 4通常在包括前列腺在内的有限数量的组织中表达，但其在前列腺中的生物学功能在很大程度上是未知的。在这项研究中，我们发现OLFM 4信使RNA在前列腺癌组织和前列腺癌细胞系中减少或检测不到。为了研究OLFM 4对前列腺癌进展的影响，我们用OLFM 4转染PC-3前列腺癌细胞以建立表达OLFM 4的PC-3细胞克隆。体外实验发现，与载体转染的对照PC-3细胞相比，表达OLFM 4的PC-3细胞克隆的增殖和侵袭力下降。此外，与注射载体转染的对照细胞的小鼠相比，注射表达OLFM 4的PC-3细胞的裸鼠表现出降低的肿瘤生长和骨侵袭和转移。机制研究表明，OLFM 4可能通过靶向组织蛋白酶D调节细胞自噬来表现出其抗癌作用，因为OLFM 4降低组织蛋白酶D蛋白水平和酶活性并减弱组织蛋白酶D诱导的癌细胞增殖。此外，在Matrigel侵袭测定中，OLFM 4的过表达废除了基质细胞衍生因子-1（SDF-1）诱导的PC-3细胞侵袭，部分通过阻断SDF-1介导的AKT磷酸化。在表达OLFM 4的PC-3细胞中的免疫共沉淀和免疫荧光染色研究证明OLFM 4与组织蛋白酶D或SDF-1之间存在直接相互作用。总之，这些结果表明OLFM 4与组织蛋白酶D和SDF-1负相互作用，并抑制前列腺癌生长和骨转移。 为了进一步研究OLFM 4基因在前列腺组织中的生理功能，我们建立并分析了OLFM 4基因敲除小鼠模型。我们已经观察到纯合OLFM 4突变小鼠在3-6个月内发生前列腺上皮增生，在10 - 12个月内发生前列腺上皮内瘤变（PIN），并且在18-20个月的小鼠前部（AP）和背侧前列腺（DLP）中发生肿瘤。 研究了相当数量的OLFM 4 +/+，没有一个人发生肿瘤，PIN的发生率在相似的时间点降低了60%。免疫组织化学染色显示OLFM 4突变（-/-）小鼠的肿瘤表达雄激素受体，但不表达神经内分泌细胞标志物突触素或基底细胞标志物34 E12和p63。与这些肿瘤的侵袭性相一致，高比例的细胞是Ki 67阳性的，并显示异常的E-钙粘蛋白表达。20月龄OLFM 4基因敲除小鼠前列腺前部（AP）的侵袭性肿瘤在HE和SMA染色中很明显。在20个月OLFM 4裸小鼠的背侧前列腺（DLP）中的局部浸润组织和肺转移的肿瘤组织被鉴定并且雄激素受体、Ki-67和E-钙粘蛋白免疫组织化学染色呈阳性。Ki-67免疫组化染色被用作前列腺上皮细胞增殖增加的指标。我们发现12和20个月OLFM 4基因敲除小鼠DLP的前列腺上皮细胞中Kki-67阳性细胞的比例显著高于对照小鼠。Ki-67阳性细胞在PIN和肿瘤中增多。这些结果表明OLFM 4基因敲除导致小鼠前列腺上皮细胞增殖能力增强。初步数据表明，早在OLFM 4缺失小鼠的前列腺组织中，AKT/GSK 3 β/β-连环蛋白信号通路的上调和细胞周期蛋白D1和c-Myc的表达增加早在3-4个月。

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.