Epigenetic identification of receptor tyrosine kinase-like orphan receptor 2 as a functional tumor suppressor inhibiting β-catenin and AKT signaling but frequently methylated in common carcinomas

Through subtraction of tumor-specific CpG methylation, we identified receptor tyrosine kinase-like orphan receptor 2 (ROR2) as a candidate tumor suppressor gene (TSG). ROR2 is a specific receptor or co-receptor for WNT5A, involved in canonical and non-canonical WNT signaling, with its role in tumorigenesis controversial. We characterized its functions and related cell signaling in common carcinomas. ROR2 was frequently silenced by promoter CpG methylation in multiple carcinomas including nasopharyngeal, esophageal, gastric, colorectal, hepatocellular, lung, and breast cancers, while no direct correlation of ROR2 and WNT5A expression was observed. Ectopic expression of ROR2 resulted in tumor suppression independent of WNT5A status, through inhibiting tumor cell growth and inducing cell cycle arrest and apoptosis. ROR2 further suppressed epithelial-mesenchymal transition and tumor cell stemness through repressing β-catenin and AKT signaling, leading to further inhibition of tumor cell migration/invasion and increased chemo-sensitivity. Thus ROR2, as an epigenetically inactivated TSG, antagonizes both β-catenin and AKT signaling in multiple tumorigenesis. Its epigenetic silencing could be a potential tumor biomarker and therapeutic target for carcinomas.     

ING1 and ING2: multifaceted tumor suppressor genes

Inhibitor of Growth 1 (ING1) was identified and characterized as a “candidate” tumor suppressor gene in 1996. Subsequently, four more genes, also characterized as “candidate” tumor suppressor genes, were identified by homology search: ING2, ING3, ING4, and ING5. The ING proteins are characterized by a high homology in their C-terminal domain, which contains a Nuclear Localization Sequence and a Plant HomeoDomain (PHD), which has a high affinity to Histone 3 tri-methylated on lysine 4 (H3K4Me3). The ING proteins have been involved in the control of cell growth, senescence, apoptosis, chromatin remodeling, and DNA repair. Within the ING family, ING1 and ING2 form a subgroup since they are evolutionarily and functionally close. In yeast, only one gene, Pho23, is related to ING1 and ING2 and possesses also a PHD. Recently, the ING1 and ING2 tumor suppressor status has been fully established since several studies have described the loss of ING1 and ING2 protein expression in human tumors and both ING1 and ING2 knockout mice were reported to have spontaneously developed tumors, B cell lymphomas, and soft tissue sarcomas, respectively. In this review, we will describe for the first time what is known about the ING1 and ING2 genes, proteins, their regulations in both human and mice, and their status in human tumors. Furthermore, we explore the current knowledge about identified functions involving ING1 and ING2 in tumor suppression pathways especially in the control of cell cycle and in genome stability.     

WDR34 is a novel TAK1-associated suppressor of the IL-1R/TLR3/TLR4-induced NF-κB activation pathway

Toll-like receptors (TLRs) act as sensors of microbial components and elicit innate immune responses. All TLR signaling pathways activate the nuclear factor-kappaB (NF-κB), which controls the expression of inflammatory cytokine genes. Transforming growth factor-β-activated kinase 1 (TAK1) is a serine/threonine protein kinase that is critically involved in the activation of NF-κB by tumor necrosis factor (TNFα), interleukin-1β (IL-1β) and TLR ligands. In this study, we identified a novel protein, WD40 domain repeat protein 34 (WDR34) as a TAK1-interacting protein in yeast two-hybrid screens. WDR34 interacted with TAK1, TAK1-binding protein 2 (TAB2), TAK1-binding protein 3 (TAB3) and tumor necrosis factor receptor-associated factor 6 (TRAF6) in overexpression and under physiological conditions. Overexpression of WDR34 inhibited IL-1β-, polyI:C- and lipopolysaccharide (LPS)-induced but not TNFα-induced NF-κB activation, whereas knockdown of WDR34 by a RNA-interference construct potentiated NF-κB activation by these ligands. Our findings suggest that WDR34 is a TAK1-associated inhibitor of the IL-1R/TLR3/TLR4-induced NF-κB activation pathway. D. Gao and R. Wang contributed equally to this work.     

Differential molecular regulation of processing and membrane expression of Type-I BMP receptors: implications for signaling

The Type-I bone morphogenetic protein receptors (BMPRs), BMPR1A and BMPR1B, present the highest sequence homology among BMPRs, suggestive of functional similitude. However, sequence elements within their extracellular domain, such as signal sequence or N-glycosylation motifs, may result in differential regulation of biosynthetic processing and trafficking and in alterations to receptor function. We show that (i) BMPR1A and the ubiquitous isoform of BMPR1B differed in mode of translocation into the endoplasmic reticulum; and (ii) BMPR1A was N-glycosylated while BMPR1B was not, resulting in greater efficiency of processing and plasma membrane expression of BMPR1A. We further demonstrated the importance of BMPR1A expression and glycosylation in ES-2 ovarian cancer cells, where (i) CRISPR/Cas9-mediated knockout of BMPR1A abrogated BMP2-induced Smad1/5/8 phosphorylation and reduced proliferation of ES-2 cells and (ii) inhibition of N-glycosylation by site-directed mutagenesis, or by tunicamycin or 2-deoxy-d-glucose treatments, reduced biosynthetic processing and plasma membrane expression of BMPR1A and BMP2-induced Smad1/5/8 phosphorylation.     

Protein kinase D inhibitor CRT0066101 suppresses bladder cancer growth in vitro and xenografts via blockade of the cell cycle at G2/M

The protein kinase D (PKD) family of proteins are important regulators of tumor growth, development, and progression. CRT0066101, an inhibitor of PKD, has antitumor activity in multiple types of carcinomas. However, the effect and mechanism of CRT0066101 in bladder cancer are not understood. In the present study, we show that CRT0066101 suppressed the proliferation and migration of four bladder cancer cell lines in vitro. We also demonstrate that CRT0066101 blocked tumor growth in a mouse flank xenograft model of bladder cancer. To further assess the role of PKD in bladder carcinoma, we examined the three PKD isoforms and found that PKD2 was highly expressed in eight bladder cancer cell lines and in urothelial carcinoma tissues from the TCGA database, and that short hairpin RNA (shRNA)-mediated knockdown of PKD2 dramatically reduced bladder cancer growth and invasion in vitro and in vivo, suggesting that the effect of the compound in bladder cancer is mediated through inhibition of PKD2. This notion was corroborated by demonstrating that the levels of phospho-PKD2 were markedly decreased in CRT0066101-treated bladder tumor explants. Furthermore, our cell cycle analysis by flow cytometry revealed that CRT0066101 treatment or PKD2 silencing arrested bladder cancer cells at the G2/M phase, the arrest being accompanied by decreases in the levels of cyclin B1, CDK1 and phospho-CDK1 (Thr161) and increases in the levels of p27^Kip1 and phospho-CDK1 (Thr14/Tyr15). Moreover, CRT0066101 downregulated the expression of Cdc25C, which dephosphorylates/activates CDK1, but enhanced the activity of the checkpoint kinase Chk1, which inhibits CDK1 by phosphorylating/inactivating Cdc25C. Finally, CRT0066101 was found to elevate the levels of Myt1, Wee1, phospho-Cdc25C (Ser216), Gadd45α, and 14-3-3 proteins, all of which reduce the CDK1-cyclin B1 complex activity. These novel findings suggest that CRT0066101 suppresses bladder cancer growth by inhibiting PKD2 through induction of G2/M cell cycle arrest, leading to the blockade of cell cycle progression.     

稳定表达绿色荧光蛋白的B16细胞株的建立及其生物学特性研究

目的建立稳定表达绿色荧光蛋白的B16黑色素瘤细胞株,并研究其生物学特性。方法转染增强型绿色荧光蛋白基因入B16细胞,经G418联合单克隆培养,筛选阳性细胞。激光共聚焦荧光显微镜观察EGFP阳性细胞形态;流式细胞仪检测细胞内EGFP基因的蛋白表达;RT-PCR检测EGFP基因的mRNA表达;皮下接种及MTF法评价细胞在体内外的生长特性。结果流式细胞仪检测表达绿色荧光蛋白的细胞阳性率为99．81％,RT-PCR检测到EGFP基因的mRNA阳性表达。生长曲线显示其体外增殖趋势与B16相似,EGFP—B16体内成瘤速度较B16慢（P〈0．05）。结论稳定表达绿色荧光蛋白的EGFP—B16细胞株成功建立,可作为作为进一步研究恶性黑色素瘤的材料。     

BAG-6, a jack of all trades in health and disease

BCL2-associated athanogene 6 (BAG-6) (also Bat-3/Scythe) was discovered as a gene product of the major histocompatibility complex class III locus. The Xenopus ortholog Scythe was first identified to act as an anti-apoptotic protein. Subsequent studies unraveled that the large BAG-6 protein contributes to a number of cellular processes, including apoptosis, gene regulation, protein synthesis, protein quality control, and protein degradation. In this context, BAG-6 acts as a multifunctional chaperone, which interacts with its target proteins for shuttling to distinct destinations. Nonetheless, as anticipated from its genomic localization, BAG-6 is involved in a variety of immunological pathways such as macrophage function and T_H1 response. Most recently, BAG-6 was identified on the plasma membrane of dendritic cells and malignantly transformed cells where it serves as cellular ligand for the activating natural killer (NK) cell receptor NKp30 triggering NK cell cytotoxicity. Moreover, target cells were found to secrete soluble variants of BAG-6 and release BAG-6 on the surface of exosomes, which inhibit or activate NK cell cytotoxicity, respectively. These data suggest that the BAG-6 antigen is an important target to shape a directed immune response or to overcome tumor-immune escape strategies established by soluble BAG-6. This review summarizes the currently known functions of BAG-6, a fascinating multicompetent protein, in health and disease.     

Domain structure and function of matrix metalloprotease 23 (MMP23): role in potassium channel trafficking

MMP23 is a member of the matrix metalloprotease family of zinc- and calcium-dependent endopeptidases, which are involved in a wide variety of cellular functions. Its catalytic domain displays a high degree of structural homology with those of other metalloproteases, but its atypical domain architecture suggests that it may possess unique functional properties. The N-terminal MMP23 pro-domain contains a type-II transmembrane domain that anchors the protein to the plasma membrane and lacks the cysteine-switch motif that is required to maintain other MMPs in a latent state during passage to the cell surface. Instead of the C-terminal hemopexin domain common to other MMPs, MMP23 contains a small toxin-like domain (TxD) and an immunoglobulin-like cell adhesion molecule (IgCAM) domain. The MMP23 pro-domain can trap Kv1.3 but not closely-related Kv1.2 channels in the endoplasmic reticulum, preventing their passage to the cell surface, while the TxD can bind to the channel pore and block the passage of potassium ions. The MMP23 C-terminal IgCAM domain displays some similarity to Ig-like C2-type domains found in IgCAMs of the immunoglobulin superfamily, which are known to mediate protein–protein and protein–lipid interactions. MMP23 and Kv1.3 are co-expressed in a variety of tissues and together are implicated in diseases including cancer and inflammatory disorders. Further studies are required to elucidate the mechanism of action of this unique member of the MMP family.     

Signaling pathways between the plasma membrane and endoplasmic reticulum calcium stores

This review discusses multiple ways in which the endoplasmic reticulum participates in and is influenced by signal transduction pathways. The endoplasmic reticulum provides a Ca2+ store that can be mobilized either by calcium-induced calcium release or by the diffusible messenger inositol 1,4,5-trisphosphate. Depletion of endoplasmic reticulum Ca2+ stores provides a signal that activates surface membrane Ca2+ channels, a process known as capacitative calcium entry. Depletion of endoplasmic reticulum stores can also signal long-term cellular responses such as gene expression and programmed cell death or apoptosis. In addition to serving as a source of cellular signals, the endoplasmic reticulum is also functionally and structurally modified by the Ca2+ and protein kinase C pathways. Elevated cytoplasmic Ca2+ causes a rearrangement and fragmentation of endoplasmic reticulum membranes. Protein kinase C activation reduces the storage capacity of the endoplasmic reticulum Ca2+ pool. In some cell types, protein kinase C inhibits capacitative calcium entry. Protein kinase C activation also protects the endoplasmic reticulum from the structural effects of high cytoplasmic Ca2+. The emerging view is one of a complex network of pathways through which the endoplasmic reticulum and the Ca2+ and protein kinase C signaling pathways interact at various levels regulating cellular structure and function.     

Crosstalk of protein kinase C ε with Smad2/3 promotes tumor cell proliferation in prostate cancer cells by enhancing aerobic glycolysis

Protein kinase C ε (PKCε) has emerged as an oncogenic protein kinase and plays important roles in cancer cell survival, proliferation, and invasion. It is, however, still unknown whether PKCε affects cell proliferation via glucose metabolism in cancer cells. Here we report a novel function of PKCε that provides growth advantages for cancer cells by enhancing tumor cells glycolysis. We found that either PKCε or Smad2/3 promoted aerobic glycolysis, expression of the glycolytic genes encoding HIF-1α, HKII, PFKP and MCT4, and tumor cell proliferation, while overexpression of PKCε or Smad3 enhanced aerobic glycolysis and cell proliferation in a protein kinase D- or TGF-β-independent manner in PC-3M and DU145 prostate cancer cells. The effects of PKCε silencing were reversed by ectopic expression of Smad3. PKCε or Smad3 ectopic expression-induced increase in cell growth was antagonized by inhibition of lactate transportation. Furthermore, interaction of endogenous PKCε with Smad2/3 was primarily responsible for phosphorylation of Ser213 in the Samd3 linker region, and resulted in Smad3 binding to the promoter of the glycolytic genes, thereby promoting cell proliferation. Forced expression of mutant Smad3 (S213A) attenuated PKCε-stimulated protein overexpression of the glycolytic genes. Thus, our results demonstrate a novel PKCε function that promotes cell growth in prostate cancer cells by increasing aerobic glycolysis through crosstalk between PKCε and Smad2/3.     

Prolactin inducible protein in cancer, fertility and immunoregulation: structure, function and its clinical implications

Prolactin inducible protein (PIP) is a 17- kDa single polypeptide chain, known by various names due to its versatile nature and function in human reproductive and immunological systems. It is expressed in several exocrine tissues such as the lacrimal, salivary, and sweat glands. Its expression is up regulated by prolactin and androgens, and estrogens down regulate it. Due to its over-expression in metastatic breast and prostate cancer, presently PIP is considered as a prognostic biomarker. Moreover, its aspartyl-proteinase nature suggests its role in tumor progression. PIP has unique features because it is small in size and plays multiple important functions. Its ability to bind potentially with CD4-T cell receptor, immunoglobulin G (IgG), actin, zinc α2-glycoprotein (ZAG), fibronectin and enamel pellicle, reveals its important biological functions. This is the first comprehensive review on the structure and functional analysis of PIP and its clinical applications. Received 04 August 2008; received after revision 09 September 2008; accepted 15 September 2008     

Antitumoral effects of 9-cis retinoic acid in adrenocortical cancer

The currently available medical treatment options of adrenocortical cancer (ACC) are limited. In our previous meta-analysis of adrenocortical tumor genomics data, ACC was associated with reduced retinoic acid production and retinoid X receptor-mediated signaling. Our objective has been to study the potential antitumoral effects of 9-cis retinoic acid (9-cisRA) on the ACC cell line NCI-H295R and in a xenograft model. Cell proliferation, hormone secretion, and gene expression have been studied in the NCI-H295R cell line. A complex bioinformatics approach involving pathway and network analysis has been performed. Selected genes have been validated by real-time qRT-PCR. Athymic nude mice xenografted with NCI-H295R have been used in a pilot in vivo xenograft model. 9-cisRA significantly decreased cell viability and steroid hormone secretion in a concentration- and time-dependent manner in the NCI-H295R cell line. Four major molecular pathways have been identified by the analysis of gene expression data. Ten genes have been successfully validated involved in: (1) steroid hormone secretion (HSD3B1, HSD3B2), (2) retinoic acid signaling (ABCA1, ABCG1, HMGCR), (3) cell-cycle damage (GADD45A, CCNE2, UHRF1), and the (4) immune response (MAP2K6, IL1R2). 9-cisRA appears to directly regulate the cell cycle by network analysis. 9-cisRA also reduced tumor growth in the in vivo xenograft model. In conclusion, 9-cisRA might represent a promising new candidate in the treatment of hormone-secreting adrenal tumors and adrenocortical cancer.     

CTGF在肺癌中的研究进展

结缔组织生长因子（CTGF／CCN2）属于即刻早期基因编码的CCN家族中的一员,参与正常细胞增殖、黏附、迁移、凋亡及新生血管形成,在创伤修复、纤维化疾病和肿瘤形成中也具有重要的作用。新近研究发现,CTGF在肺癌组织中表达较正常肺组织降低,与肺癌的发生、发展密切相关,被视为肺癌转移和预后的评价指标。本文简要介绍了CTGF的结构、功能及在其肺癌中的研究进展,以期为肺癌的临床治疗提供新的思路。     

KIF1Bβ transports dendritically localized mRNPs in neurons and is recruited to synapses in an activity-dependent manner

KIF1Bβ is a kinesin-like, microtubule-based molecular motor protein involved in anterograde axonal vesicular transport in vertebrate and invertebrate neurons. Certain KIF1Bβ isoforms have been implicated in different forms of human neurodegenerative disease, with characterization of their functional integration and regulation in the context of synaptic signaling still ongoing. Here, we characterize human KIF1Bβ (isoform NM015074), whose expression we show to be developmentally regulated and elevated in cortical areas of the CNS (including the motor cortex), in the hippocampus, and in spinal motor neurons. KIF1Bβ localizes to the cell body, axon, and dendrites, overlapping with synaptic-vesicle and postsynaptic-density structures. Correspondingly, in purified cortical synaptoneurosomes, KIF1Bβ is enriched in both pre- and postsynaptic structures, forming detergent-resistant complexes. Interestingly, KIF1Bβ forms RNA–protein complexes, containing the dendritically localized Arc and Calmodulin mRNAs, proteins previously shown to be part of RNA transport granules such as Purα, FMRP and FXR2P, and motor protein KIF3A, as well as Calmodulin. The interaction between KIF1Bβ and Calmodulin is Ca⁺²-dependent and takes place through a domain mapped at the carboxy-terminal tail of the motor. Live imaging of cortical neurons reveals active movement by KIF1Bβ at dendritic processes, suggesting that it mediates the transport of dendritically localized mRNAs. Finally, we show that synaptic recruitment of KIF1Bβ is activity-dependent and increased by stimulation of metabotropic or ionotropic glutamate receptors. The activity-dependent synaptic recruitment of KIF1Bβ, its interaction with Ca²⁺ sensor Calmodulin, and its new role as a dendritic motor of ribonucleoprotein complexes provide a novel basis for understanding the concerted co-ordination of motor protein mobilization and synaptic signaling pathways.