The novel NLR-related protein NWD1 is associated with prostate cancer progression and impacts androgen receptor signaling

Characterization of NWD1; a novel NLR-related protein and further correlate it as a putative Prostate Cancer marker. NLRs (NACHT and Leucine Rich Repeat domain containing proteins) constitute a major subfamily of innate immunity proteins mostly acting as cytosolic pattern recognition receptors (PRRs), involved in the detection of cytoplasmic pathogen-associated molecular patterns (PAMPs) and endogenous danger signals. The recognition of the signals initiates a variety of host defense pathways through the activation of NF-kappaB, stress kinases, interferon response factors (IRFs) and/or inflammatory caspases. Despite the importance in host immune response, deregulation on NLR activities has been described in a variety of maladies, including chronic inflammation and cancer predisposition. For instance, NOD1 was one of the first NLR members shown to possibly play a role in tumorigenesis, since NOD1 stimulation induces apoptosis in MCF-7 breast carcinoma cells, and NOD1-/- MCF-7 cells generate larger tumors after injection in SCID mice. Mice deficient in NLR member NLRP3, and/or its interacting partners ASC or caspase-1, are also shown to be more susceptible to colitis-associated cancer (CAC). Polymorphisms along NLR genes NOD1 and NOD2 have also been correlated with altered cancer risk. NOD1 SNPs have been associated with gastric cancer, lymphoma, ovarian, prostate, and lung cancer due to the recognition of H. pylori (etiologic agent in gastric cancer and MALT lymphoma), C. trachomatis (putative etiologic agent in ovarian cancer), P. acnes (possible causative agent in PCa) and C. pneumonia (plausible etiological agent in lung cancer) as NOD1 ligands. Particularly, NOD1 and NOD2 have been shown to be fully operative in prostate epithelial cells and, in cooperation with TLRs, may elicit immune response during PCa progression. To access a tissue-specific gene expression profile for NWD1, quantitative PCR analysis was performed using a normalized cDNA panel derived from 48 human tissues (TissueScan Tissue qPCR Array, Origene). Two apparently independent expression patterns were detected, respectively related to neurological related organs (brain, pituitary and retina) and male reproductive system (prostate, epididymis and testis), where the highest mRNA levels was observed in prostate tissue. Results were confirmed using a second set of NWD1-specific qPCR primers (data not shown). Moreover, a similar expression pattern was virtually observed using the SAGE database from the Cancer Genome Anatomy Project (CGAP) of the National Cancer Institute (not shown). Comparative gene expression microarray data was analyzed systematically by Ingenuity Pathway Analysis (IPA) to set up potential networks based on NWD1 regulated genes. The eventual participation of NWD1 in signaling transduction pathways was further examined by microarray analysis (Human WG-6v3 Expression BeadChip, Illumina) comparing the expression profile of PPC-1 cells lacking NWD1 expression (sh184 & sh3922) versus control cells (shGFP). According to the differential expression profile that was generated and analyzed by the Ingenuity PathwaysTM software (IPA version 7.1, Ingenuity Systems), NWD1 is presumably associated with biological networks related, for instance, to tissue morphology, organogenesis, cancer and neurological diseases .

NWD1的功能表征：一种新型NLR相关蛋白，并进一步验证其作为潜在前列腺癌标志物的可能性。NLR（含NACHT结构域与富亮氨酸重复序列结构域的蛋白）是先天免疫蛋白的一个主要亚家族，主要作为胞质模式识别受体（PRRs），参与识别胞质内的病原体相关分子模式（PAMPs）与内源性危险信号。对上述信号的识别可通过激活NF-κB、应激激酶、干扰素应答因子（IRFs）以及炎性半胱天冬酶等，启动多条宿主防御通路。尽管NLR在宿主免疫应答中发挥关键作用，但已有研究表明，多种疾病（包括慢性炎症与肿瘤易感性）中均存在NLR活性失调的情况。例如，NOD1是首批被发现可能参与肿瘤发生的NLR家族成员之一：研究发现，激活NOD1可诱导MCF-7乳腺癌细胞发生凋亡，而NOD1基因敲除的MCF-7细胞接种至重症联合免疫缺陷（SCID）小鼠体内后可形成更大的肿瘤。此外，NLR家族成员NLRP3或其互作蛋白ASC、半胱天冬酶-1（caspase-1）基因敲除小鼠也被证实更易罹患结肠炎相关癌症（CAC）。NLR家族基因NOD1与NOD2的基因多态性也与癌症风险改变存在关联。NOD1的单核苷酸多态性（SNPs）与胃癌、淋巴瘤、卵巢癌、前列腺癌及肺癌均存在关联，这是因为NOD1可识别作为其配体的多种病原体：包括幽门螺杆菌（胃癌与黏膜相关淋巴组织淋巴瘤的致病原）、沙眼衣原体（卵巢癌的潜在致病原）、痤疮丙酸杆菌（前列腺癌的可能致病原）以及肺炎衣原体（肺癌的疑似致病原）。尤其值得注意的是，已有研究证实NOD1与NOD2在前列腺上皮细胞中可完全发挥功能，并可与Toll样受体（TLRs）协同，在前列腺癌（PCa）进展过程中触发免疫应答。为获取NWD1的组织特异性基因表达谱，研究团队使用源自48种人体组织的标准化cDNA组合（Origene公司TissueScan组织qPCR芯片）开展了定量PCR分析。研究检测到两种明显独立的表达模式：分别与神经系统相关器官（大脑、垂体与视网膜）以及男性生殖系统（前列腺、附睾与睾丸）相关，其中前列腺组织中的NWD1 mRNA水平最高。该结果通过另一组NWD1特异性qPCR引物得到了验证（数据未展示）。此外，通过美国国家癌症研究所癌症基因组解剖项目（CGAP）的基因表达系列分析（SAGE）数据库分析，也得到了相似的表达模式（数据未展示）。研究人员通过IPA（Ingenuity Pathway Analysis）对比较基因表达芯片数据进行了系统分析，以构建NWD1调控基因的潜在调控网络。为进一步探究NWD1是否参与信号转导通路，研究团队通过Illumina公司Human WG-6v3表达微珠芯片开展了芯片分析，对比了NWD1基因敲低的PPC-1细胞（sh184与sh3922）与对照细胞（shGFP）的表达谱。通过Ingenuity PathwaysTM软件（IPA 7.1版本，Ingenuity Systems公司）对差异表达谱进行分析后发现，NWD1大概率与多个生物调控网络相关，例如组织形态发生、器官发育、癌症以及神经系统疾病相关网络。