microRNAs as oncogenes and tumor suppressors

microRNAs (miRNAs) are a new class of non-protein-coding, endogenous, small RNAs. They are important regulatory molecules in animals and plants. miRNA regulates gene expression by translational repression, mRNA cleavage, and mRNA decay initiated by miRNA-guided rapid deadenylation. Recent studies show that some miRNAs regulate cell proliferation and apoptosis processes that are important in cancer formation. By using multiple molecular techniques, which include Northern blot analysis, real-time PCR, miRNA microarray, up- or down-expression of specific miRNAs, it was found that several miRNAs were directly involved in human cancers, including lung, breast, brain, liver, colon cancer, and leukemia. In addition, some miRNAs may function as oncogenes or tumor suppressors. More than 50% of miRNA genes are located in cancer-associated genomic regions or in fragile sites, suggesting that miRNAs may play a more important role in the pathogenesis of a limited range of human cancers than previously thought. Overexpressed miRNAs in cancers, such as mir-17-92, may function as oncogenes and promote cancer development by negatively regulating tumor suppressor genes and/or genes that control cell differentiation or apoptosis. Underexpressed miRNAs in cancers, such as let-7, function as tumor suppressor genes and may inhibit cancers by regulating oncogenes and/or genes that control cell differentiation or apoptosis. miRNA expression profiles may become useful biomarkers for cancer diagnostics. In addition, miRNA therapy could be a powerful tool for cancer prevention and therapeutics.     

Apaf-1 and caspase-9 do not act as tumor suppressors in myc-induced lymphomagenesis or mouse embryo fibroblast transformation

Based on experiments with cultured fibroblasts, the apoptosis regulators caspase-9 and Apaf-1 are hypothesized to function as tumor suppressors. To investigate their in vivo role in lymphomagenesis, an IgH enhancer-driven c-myc transgene was crossed onto Apaf-1(-/-) and caspase-9(-/-) mice. Due to perinatal lethality, Emu-myc transgenic Apaf-1(-/-) or caspase-9(-/-) fetal liver cells were used to reconstitute lethally irradiated recipient mice. Surprisingly, no differences were seen in rate, incidence, or severity of lymphoma with loss of Apaf-1 or caspase-9, and Apaf-1 was not a critical determinant of anticancer drug sensitivity of c-myc-induced lymphomas. Moreover, loss of Apaf-1 did not promote oncogene-induced transformation of mouse embryo fibroblasts. Thus, Apaf-1 and caspase-9 do not suppress c-myc-induced lymphomagenesis and embryo fibroblast transformation.     

肿瘤生长抑制因子—血管抑素和内皮抑素

血管生成是肿瘤生长转移过程中的一个关键环节,因此控制血管生成成为抑制肿瘤生长的重要途径之一。目前已发现了许多血管生成抑制因子,尤以血管抑素和内皮抑素最为引人瞩目。综述了两种肿瘤生长抑制因子的发现、分子结构、生物学活性等,尤其侧重于它们抗肿瘤作用的实验研究。血管抑素与内皮抑素的发现与研究为恶性肿瘤的治疗开辟了新的道路。     

TRAIL death receptors as tumor suppressors and drug targets

Various ways of targeting TRAIL-death receptors for the treatment of a diverse set of malignancies are being explored in ongoing clinical trials. Recent data of ours and others suggest that loss of the only death signaling receptor in mice (TRAIL-R) is associated with susceptibility to various stages of lymphomagenesis and carcinogenesis, perhaps in a complex cell- and model-specific manner (1,2). Myc-overexpressing B cell lymphomas with an intact TRAIL-R locus displayed a number of gene expression changes indicating resistance to TRAIL-R signaling. Herein we show some data on the use of recombinant human TRAIL (rhTRAIL) and γ-radiation (10 Gy) in combination in an autochthonous mouse model for hepatocellular carcinoma. As cell death signaling through the death receptors is evolutionary conserved from zebra fish to man, novel genetically engineered mouse tumor models may prove useful in establishing in vivo models that excel our fundamental understanding of resistance to TRAIL-death receptor signaling, off-target effects from TRAIL-death receptor targeting compounds and help in identifying a clinically cogent rationale for efficient targeting of TRAIL death receptors in patients. Once established, mouse tumor models may prove to be a useful tool in understanding TRAIL-death receptor signaling.     

Mutations in eukaryotic release factors 1 and 3 act as general nonsense suppressors in Drosophila

In a screen for suppressors of the Drosophila wingless(PE4) nonsense allele, we isolated mutations in the two components that form eukaryotic release factor. eRF1 and eRF3 comprise the translation termination complex that recognizes stop codons and catalyzes the release of nascent polypeptide chains from ribosomes. Mutations disrupting the Drosophila eRF1 and eRF3 show a strong maternal-effect nonsense suppression due to readthrough of stop codons and are zygotically lethal during larval stages. We tested nonsense mutations in wg and in other embryonically acting genes and found that different stop codons can be suppressed but only a subset of nonsense alleles are subject to suppression. We suspect that the context of the stop codon is significant: nonsense alleles sensitive to suppression by eRF1 and eRF3 encode stop codons that are immediately followed by a cytidine. Such suppressible alleles appear to be intrinsically weak, with a low level of readthrough that is enhanced when translation termination is disrupted. Thus the eRF1 and eRF3 mutations provide a tool for identifying nonsense alleles that are leaky. Our findings have important implications for assigning null mutant phenotypes and for selecting appropriate alleles to use in suppressor screens.     

Adenovirus type 5 E1A gene products act as transformation suppressors of the neu oncogene.

The adenovirus type 5 early region 1A (E1A) gene was introduced into neu-transformed B104-1-1 cells. Cells that expressed E1A possessed reduced transforming activity in vitro and reduced tumorigenicity in nude mice. These results demonstrate that the E1A gene products can act negatively to suppress the transformed phenotype in neu-transformed cells.     

GABAB receptor complex as a potential target for tumor therapy

γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the vertebrate central nervous system. Metabotropic GABA(B) receptors are heterodimeric G-protein-coupled receptors (GPCRs) consisting of GABA(B1) and GABA(B2) subunits. The intracellular C-terminal domains of GABA(B) receptors are involved in heterodimerization, oligomerization, and association with other proteins, which results in a large receptor complex. Multiple splice variants of the GABA(B1) subunit have been identified in which GABA(B1a) and GABA(B1b) are the most abundant isoforms in the nervous system. Isoforms GABA(B1c) through GABA(B1n) are minor isoforms and are detectable only at mRNA levels. Some of the minor isoforms have been detected in peripheral tissues and encode putative soluble proteins with C-terminal truncations. Interestingly, increased expression of GABA(B) receptors has been detected in several human cancer cells and tissues. Moreover, GABA(B) receptor agonist baclofen inhibited tumor growth in rat models. GABA(B) receptor activation not only induces suppressing the proliferation and migration of various human tumor cells but also results in inactivation of CREB (cAMP-responsive element binding protein) and ERK in tumor cells. Their structural complexity makes it possible to disrupt the functions of GABA(B) receptors in various ways, raising GABA(B) receptor diversity as a potential therapeutic target in some human cancers.     

Tyrosine phosphatases as a superfamily of tumor suppressors in colorectal cancer

Phosphorylation and dephosphorylation processes catalyzed by numerous kinases and phosphorylases are essential for cell homeostasis and may lead to disturbances in a variety of vital cellular pathways, such as cell proliferation and differentiation, and thus to complex diseases including cancer. As over 80 % of all oncogenes encode protein tyrosine kinases (PTKs), protein tyrosine phosphatases (PTPs), which can reverse the effects of tyrosine kinases, are very important tumor suppressors. Alterations in tyrosine kinase and phosphatase genes including point mutations, changes in epigenetic regulation, as well as chromosomal aberrations involving regions critical to these genes, are frequently observed in a variety of cancers. Colorectal cancer (CRC) is one of the most common cancers in humans. CRCs occur in a familial (about 15 % of all cases), hereditary (about 5%) and sporadic (almost 75-80 %) form. As genetic-environmental interrelations play an important role in the susceptibility to sporadic forms of CRCs, many studies are focused on genetic alterations in such tumors. Mutational analysis of the tyrosine phosphatome in CRCs has identified somatic mutations in PTPRG, PTPRT, PTPN3, PTPN13 and PTPN14. The majority of these mutations result in a loss of protein function. Also, alterations in the expression of these genes, such as decreased expression of PTPRR, PTPRO, PTPRG and PTPRD, mediated by epigenetic mechanisms have been observed in a variety of tumors. Since cancer is a social and global problem, there will be a growing number of studies on alterations in the candidate cancer genes, including protein kinases and phosphatases, to determine the origin, biology and potential pathways for targeted anticancer therapy.     

Nuclear tumor suppressors in space and time

Numerous studies have identified key binding partners and functional activities of nuclear tumor-suppressor proteins such as the retinoblastoma protein, p53 and BRCA1. Historically, less attention has been given to the subnuclear locations of these proteins. Here, we describe several recent studies that promote the view that regulated association with subcompartments of the nucleus is inherent to tumor-suppressor function.     

Accessorizing and anchoring the LINC complex for multifunctionality

A migrating cell must establish front-to-back polarity in order to move. In this issue, Juanes-Garcia et al. (2015. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201407059) report that a short serine-rich motif in nonmuscle myosin IIB is required to establish the cell’s rear. This motif represents a new paradigm for what determines directional cell migration.Directed cell movement is instrumental for organismal development, immune responses, and the progression of diseases, such as cancer (Gardel et al., 2010). To achieve directed movement, an individual cell must establish front-to-back polarity, where there is coordinated protrusion of its front and retraction of its back (Fig. 1 A; Vicente-Manzanares et al., 2007). How polymerizing actin filaments drive protrusion of the front is understood in exquisite detail (Pollard and Borisy, 2003; Pollard, 2007). The mechanisms defining how actin filament contraction defines the back of the cell (Yam et al., 2007) have been more difficult to elucidate. Contraction of actin filaments in crawling cells is driven by nonmuscle myosin II (NMII; Vicente-Manzanares et al., 2009). NMII has three isoforms, NMII-A, NMII-B, and NMII-C, all of which can bind and contract actin filaments to generate force. Importantly, NMII-A and NMII-B have different cellular localizations, which could drive their functions (Kolega, 1998; Vicente-Manzanares et al., 2007). NMII-A localizes primarily to the front, protrusive edge and is required for adhesion maturation. In contrast, NMII-B localizes behind NMII-A, primarily to large and stable actin stress fibers in the middle and back of the cell (Kolega, 1998; Vicente-Manzanares et al., 2007). NMII-B is required for front-to-back polarity, as cells lacking NMII-B lose large stress fibers and fail to define their rear (Vicente-Manzanares et al., 2007). The major question of what drives the polarized localization of NMII-B is unknown. In this issue, Juanes-Garcia et al. report that a short serine-rich motif in NMII-B is responsible for both its localization and the establishment of front-to-back cellular polarity.Open in a separate windowFigure 1.Nonmuscle myosins in cell migration. (A) Schematic showing a top view of a crawling cell. The front of the cell is protruding (top arrow), and the back of the cell is retracting (bottom arrow). The protrusion of the edge is driven by polymerization of actin filament networks in the lamellipodium (gray hash marks). NMII-containing stress fibers (SF, dark blue lines) are assembled behind the lamellipodium. SFs are connected to focal adhesions (gray ovals) either directly or indirectly through non-NMII–containing actin bundles (Dorsal SF, light blue lines; Naumanen et al., 2008). Moving away from the cell’s front, there is a decreasing and increasing gradient of NMII-A (red wedge) and NMII-B (green wedge), respectively (Kolega, 1998). (B) Schematic of NMII-A and NMII-B isoforms. A single NMII molecule is a hexamer of two heavy chains (i.e., NMII-A, NMII-B, or NMIIC), two regulatory light chains, and two essential light chains (Vicente-Manzanares et al., 2009). The overall structure of NMII-A and NMII-B molecules is similar, with two motor domains, a coiled-coil rod domain, and a short nonhelical tail domain. The serine-rich motif is unique to NMII-B, and the role for this motif in SF contraction and the ability of the cell to apply forces to its environment are yet to be determined.Though NMII-A and NMII-B are genetically and structurally very similar, Juanes-Garcia et al. (2015) identified a serine-rich sequence (SFSSSRS) in the C terminus of NMII-B (Fig. 1 B). The authors effectively used cells depleted of NMII-B (Vicente-Manzanares et al., 2007) to test the role of this serine motif in front-to-back polarity. Although expression of wild-type NMII-B rescued front-to-back polarity, expressing NMII-B lacking the serine motif did not. Interestingly, simply inserting the serine-rich motif from NMII-B into NMII-A (NMII-A5S) conferred the ability to rescue front-to-back polarity. In addition, NMII-A5S did not localize to the front of the cell or play a role in adhesion maturation like wild-type NMII-A. Mass spectrometry analysis revealed three of the residues in the serine motif of NMII-B were phosphorylated in cells, and one of these, serine 1935, was found to be crucial for the wild-type kinetics and localization of NMII-B. A phosphomimetic point mutation, S1935D, failed to rescue front-to-back polarity in NMII-B–depleted cells. In contrast, expression of the nonphosphorylatable mutant, S1935A, localized normally to large actin stress fibers and did rescue front-to-back polarity.To provide further evidence that serine 1935 is a regulatory element of front-to-back polarity, Juanes-Garcia et al. (2015) investigated the role of PKC, which acts upstream of NMII in cell polarization (Gomes et al., 2005; Even-Faitelson and Ravid, 2006), in NMII-B–generated stable actin bundles. Cells expressing constitutively active PKC produced isotropic protrusions at the perimeter of the cell, while also failing to produce large, stable NMII-B decorated actin bundles. This isotropic protrusive phenotype was blocked when nonphosphorylatable NMII-B S1935A was expressed in cells but not with wild type or S1935D. Thus, PKC was implicated as the likely upstream regulator of NMII-B activity, which negatively regulates stable actin stress fiber formation by phosphorylating NMII-B at serine 1935. Taken together, the data presented strongly suggest that a small regulatory motif on NMII-B controls cellular front-to-back polarity in migrating cells.The findings presented in this issue by Juanes-Garcia et al. (2015) shine a bright spotlight on a family of motors that has already taken “center stage” in cellular research (Vicente-Manzanares et al., 2009). Some exciting new questions as to how NMII-B functions in the establishment of asymmetric cellular shape and function can now be addressed, including but clearly not limited to: How does the unique enzymatic activity of NMII-B’s motor domain synergize with the serine motif to drive front-to-back polarity (Billington et al., 2013)? What are the structural and dynamic implications for homo- and/or hetero-NMII filament formation (Ricketson et al., 2010; Beach et al., 2014; Shutova et al., 2014)? Does the NMII-B serine motif play a role in the establishment of more complex 3D cellular shapes? Is the serine motif required for directional cell migration through physiological environments?     

The actin cytoskeleton-associated protein zyxin acts as a tumor suppressor in Ewing tumor cells

Changes in cell architecture, essentially linked to profound cytoskeleton rearrangements, are common features accompanying cell transformation. Supporting the involvement of the microfilament network in tumor cell behavior, several actin-binding proteins, including zyxin, a potential regulator of actin polymerization, may play a role in oncogenesis. In this work, we investigate the status of zyxin in Ewing tumors, a family of pediatric malignancies of bone and soft tissues, which are mainly associated with a t(11;22) chromosomal translocation encoding the EWS-FLI1 oncoprotein. We observe that EWS-FLI1-transformed murine fibroblasts, as well as human Ewing tumor-derived SK-N-MC cells, exhibit a complete disruption of their actin cytoskeleton, retaining very few stress fibers, focal adhesions and cell-to-cell contacts. We show that within these cells, zyxin is expressed at very low levels and remains diffusely distributed throughout the cytoplasm, instead of concentrating in actin-rich dynamic structures. We demonstrate that zyxin gene transfer into EWS-FLI1-transformed fibroblasts elicits reconstitution of zyxin-rich focal adhesions and intercellular junctions, dramatic reorganization of the actin cytoskeleton, decreased cell motility, inhibition of anchorage-independent growth and impairment of tumor formation in athymic mice. We observe similar phenotypic changes after zyxin gene transfer in SK-N-MC cells, suggesting that zyxin has tumor suppressor activity in Ewing tumor cells.     

Characterization of the human tumor suppressors TIG3 and HRASLS2 as phospholipid-metabolizing enzymes

Tazarotene-induced protein 3 (TIG3) and HRAS-like suppressor family 2 (HRASLS2) exhibit tumor-suppressing activities and belong to the lecithin retinol acyltransferase (LRAT) protein family. Since Ca²⁺-independent N-acyltransferase and H-rev107 (another tumor suppressor), both of which are members of the LRAT family, have been recently reported to possess catalytic activities related to phospholipid metabolism, we examined possible enzyme activities of human TIG3 and HRASLS2 together with human H-rev107. The purified recombinant proteins of TIG3, HRASLS2, and H-rev107 functioned as phospholipase (PL) A_1/2 in a Ca²⁺-independent manner with maximal activities of 0.53, 0.67, and 2.57 μmol/min/mg of protein, respectively. The proteins were active with various phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs), and for most of substrates the PLA₁ activity was much higher than the PLA₂ activity. In addition, HRASLS2 catalyzed N-acylation of PE to form N-acyl-PE and O-acylation of lyso PC to form PC. TIG3 and H-rev107 catalyzed the N-acylation and O-acylation at relatively low rates. Moreover, these three proteins showed different expression profiles in human tissues. These results suggest that the tumor suppressors TIG3, HRASLS2 and H-rev107 are involved in the phospholipid metabolism with different physiological roles.     

Identification of LINC01615 as potential metastasis-related long noncoding RNA in hepatocellular carcinoma

Hepatocellular carcinoma is one of the most prevalent and fatal cancers. Studying the long noncoding RNA (lncRNA) alterations in hepatocellular carcinoma may lead to new therapeutic strategies. We checked whether there were correlations between The Cancer Genome Atlas expression profiles of the differentially expressed lncRNAs and their DNA methylation status or the copy number variations for hepatocellular carcinoma. We obtained 41 lncRNAs that were differentially expressed between tumor and normal samples, and their DNA methylation status was negatively correlated with the expression levels. We identified five lncRNAs that were recurrently amplified or deleted in tumor samples, but none of them were associated with the messenger RNA (mRNA) expression levels. To obtain the biological function of these lncRNAs, the coexpressed mRNAs in the hepatocellular carcinoma were figured out. A total of 10 lncRNAs were highly correlated with at least one gene. Six out of the ten lncRNAs were already known to be related with cancer previously. LINC01615 had 72 coexpressed genes, and we carried out the gene ontology (GO) term enrichment for these protein-coding genes. The results suggested that these lncRNAs were associated with extracellular matrix organization. To summarize, we identified 41 potentially cancer-related lncRNAs. In particular, we proposed that LINC01615 potentially affected the extracellular matrix and had further impacts on the metastasis of hepatocellular carcinoma.     

Identification of a tripartite import signal in the Ewing Sarcoma protein (EWS)

The Ewing Sarcoma (EWS) protein is a ubiquitously expressed RNA processing factor that localises predominantly to the nucleus. However, the mechanism through which EWS enters the nucleus remains unclear, with differing reports identifying three separate import signals within the EWS protein. Here we have utilized a panel of truncated EWS proteins to clarify the reported nuclear localisation signals. We describe three C-terminal domains that are important for efficient EWS nuclear localization: (1) the third RGG-motif; (2) the last 10 amino acids (known as the PY-import motif); and (3) the zinc-finger motif. Although these three domains are involved in nuclear import, they are not independently capable of driving the efficient import of a GFP-moiety. However, collectively they form a complex tripartite signal that efficiently drives GFP-import into the nucleus. This study helps clarify the EWS import signal, and the identification of the involvement of both the RGG- and zinc-finger motifs has wide reaching implications.