KSHV MicroRNAs Mediate Cellular Transformation and Tumorigenesis by Redundantly Targeting Cell Growth and Survival Pathways

Kaposi''s sarcoma-associated herpesvirus (KSHV) is causally linked to several human cancers, including Kaposi''s sarcoma, primary effusion lymphoma and multicentric Castleman''s disease, malignancies commonly found in HIV-infected patients. While KSHV encodes diverse functional products, its mechanism of oncogenesis remains unknown. In this study, we determined the roles KSHV microRNAs (miRs) in cellular transformation and tumorigenesis using a recently developed KSHV-induced cellular transformation system of primary rat mesenchymal precursor cells. A mutant with a cluster of 10 precursor miRs (pre-miRs) deleted failed to transform primary cells, and instead, caused cell cycle arrest and apoptosis. Remarkably, the oncogenicity of the mutant virus was fully restored by genetic complementation with the miR cluster or several individual pre-miRs, which rescued cell cycle progression and inhibited apoptosis in part by redundantly targeting IκBα and the NF-κB pathway. Genomic analysis identified common targets of KSHV miRs in diverse pathways with several cancer-related pathways preferentially targeted. These works define for the first time an essential viral determinant for KSHV-induced oncogenesis and identify NF-κB as a critical pathway targeted by the viral miRs. Our results illustrate a common theme of shared functions with hierarchical order among the KSHV miRs.     

Kaposi's Sarcoma Herpesvirus K15 Protein Contributes to Virus-Induced Angiogenesis by Recruiting PLCγ1 and Activating NFAT1-dependent RCAN1 Expression

Kaposi''s Sarcoma (KS), caused by Kaposi''s Sarcoma Herpesvirus (KSHV), is a highly vascularised angiogenic tumor of endothelial cells, characterized by latently KSHV-infected spindle cells and a pronounced inflammatory infiltrate. Several KSHV proteins, including LANA-1 (ORF73), vCyclin (ORF72), vGPCR (ORF74), vIL6 (ORF-K2), vCCL-1 (ORF-K6), vCCL-2 (ORF-K4) and K1 have been shown to exert effects that can lead to the proliferation and atypical differentiation of endothelial cells and/or the secretion of cytokines with angiogenic and inflammatory properties (VEGF, bFGF, IL6, IL8, GROα, and TNFβ). To investigate a role of the KSHV K15 protein in KSHV-mediated angiogenesis, we carried out a genome wide gene expression analysis on primary endothelial cells infected with KSHV wildtype (KSHVwt) and a KSHV K15 deletion mutant (KSHVΔK15). We found RCAN1/DSCR1 (Regulator of Calcineurin 1/Down Syndrome critical region 1), a cellular gene involved in angiogenesis, to be differentially expressed in KSHVwt- vs KSHVΔK15-infected cells. During physiological angiogenesis, expression of RCAN1 in endothelial cells is regulated by VEGF (vascular endothelial growth factor) through a pathway involving the activation of PLCγ1, Calcineurin and NFAT1. We found that K15 directly recruits PLCγ1, and thereby activates Calcineurin/NFAT1-dependent RCAN1 expression which results in the formation of angiogenic tubes. Primary endothelial cells infected with KSHVwt form angiogenic tubes upon activation of the lytic replication cycle. This effect is abrogated when K15 is deleted (KSHVΔK15) or silenced by an siRNA targeting the K15 expression. Our study establishes K15 as one of the KSHV proteins that contribute to KSHV-induced angiogenesis.     

Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) Induces the Oncogenic miR-17-92 Cluster and Down-Regulates TGF-β Signaling

KSHV is a DNA tumor virus that causes Kaposi’s sarcoma. Upon KSHV infection, only a limited number of latent genes are expressed. We know that KSHV infection regulates host gene expression, and hypothesized that latent genes also modulate the expression of host miRNAs. Aberrant miRNA expression contributes to the development of many types of cancer. Array-based miRNA profiling revealed that all six miRNAs of the oncogenic miR-17-92 cluster are up-regulated in KSHV infected endothelial cells. Among candidate KSHV latent genes, we found that vFLIP and vCyclin were shown to activate the miR-17-92 promoter, using luciferase assay and western blot analysis. The miR-17-92 cluster was previously shown to target TGF-β signaling. We demonstrate that vFLIP and vCyclin induce the expression of the miR-17-92 cluster to strongly inhibit the TGF-β signaling pathway by down-regulating SMAD2. Moreover, TGF-β activity and SMAD2 expression were fully restored when antagomirs (inhibitors) of miR-17-92 cluster were transfected into cells expressing either vFLIP or vCyclin. In addition, we utilized viral genetics to produce vFLIP or vCyclin knock-out viruses, and studied the effects in infected TIVE cells. Infection with wildtype KSHV abolished expression of SMAD2 protein in these endothelial cells. While single-knockout mutants still showed a marked reduction in SMAD2 expression, TIVE cells infected by a double-knockout mutant virus were fully restored for SMAD2 expression, compared to non-infected TIVE cells. Expression of either vFLIP or vCycIin was sufficient to downregulate SMAD2. In summary, our data demonstrate that vFLIP and vCyclin induce the oncogenic miR-17-92 cluster in endothelial cells and thereby interfere with the TGF-β signaling pathway. Manipulation of the TGF-β pathway via host miRNAs represents a novel mechanism that may be important for KSHV tumorigenesis and angiogenesis, a hallmark of KS.     

BRCA1: cell cycle checkpoint, genetic instability, DNA damage response and cancer evolution

Germline mutations of the breast cancer associated gene 1 (BRCA1) predispose women to breast and ovarian cancers. BRCA1 is a large protein with multiple functional domains and interacts with numerous proteins that are involved in many important biological processes/pathways. Mounting evidence indicates that BRCA1 is involved in all phases of the cell cycle and regulates orderly events during cell cycle progression. BRCA1 deficiency, consequently causes abnormalities in the S-phase checkpoint, the G₂/M checkpoint, the spindle checkpoint and centrosome duplication. The genetic instability caused by BRCA1 deficiency, however, also triggers cellular responses to DNA damage that blocks cell proliferation and induces apoptosis. Thus BRCA1 mutant cells cannot develop further into full-grown tumors unless this cellular defense is broken. Functional analysis of BRCA1 in cell cycle checkpoints, genome integrity, DNA damage response (DDR) and tumor evolution should benefit our understanding of the mechanisms underlying BRCA1 associated tumorigenesis, as well as the development of therapeutic approaches for this lethal disease.     

Nutlin-3 induces apoptosis,disrupts viral latency and inhibits expression of angiopoietin-2 in Kaposi sarcoma tumor cells

Kaposi sarcoma (KS) tumors often contain a wild-type p53. However, the function of this tumor suppressor in KS tumor cells is inhibited by both MDM2 and latent nuclear antigen (LANA) of Kaposi sarcoma-associated herpes virus (KSHV). Here, we report that MDM2 antagonist Nutlin-3 efficiently reactivates p53 in telomerase-immortalized human umbilical vein endothelial cells (TIVE) that had been malignantly transformed by KSHV as well as in KS tumor cells. Reactivation of p53 results in a G₁ cell cycle arrest, leading to inhibition of proliferation and apoptosis. Nutlin-3 inhibits the growth of “KS-like” tumors resulting from xenografted TIVE-KSHV cells in nude mice. In addition, Nutlin-3 strongly inhibits expression of the pro-angiogenic and pro-inflammatory cytokine angiopoietin-2 (Ang-2). It also disrupts viral latency by inducing expression of KSHV lytic genes. These results suggest that Nutlin-3 might serve as a novel therapy for KS.     

Wilms’ Tumor Gene 1 (WT1) Silencing Inhibits Proliferation of Malignant Peripheral Nerve Sheath Tumor sNF96.2 Cell Line

Wilms’ tumor gene 1 (WT1) plays complex roles in tumorigenesis, acting as tumor suppressor gene or an oncogene depending on the cellular context. WT1 expression has been variably reported in both benign and malignant peripheral nerve sheath tumors (MPNSTs) by means of immunohistochemistry. The aim of the present study was to characterize its potential pathogenetic role in these relatively uncommon malignant tumors. Firstly, immunohistochemical analyses in MPNST sNF96.2 cell line showed strong WT1 staining in nuclear and perinuclear areas of neoplastic cells. Thus, we investigated the effects of silencing WT1 by RNA interference. Through Western Blot analysis and proliferation assay we found that WT1 knockdown leads to the reduction of cell growth in a time- and dose-dependent manner. siWT1 inhibited proliferation of sNF96.2 cell lines likely by influencing cell cycle progression through a decrease in the protein levels of cyclin D1 and inhibition of Akt phosphorylation compared to the control cells. These results indicate that WT1 knockdown attenuates the biological behavior of MPNST cells by decreasing Akt activity, demonstrating that WT1 is involved in the development and progression of MPNSTs. Thus, WT1 is suggested to serve as a potential therapeutic target for MPNSTs.     

Dephosphorylation of CaMKII at T253 controls the metaphase–anaphase transition

Calcium/calmodulin-stimulated protein kinase II (CaMKII) is a multi-functional serine/threonine protein kinase that controls a range of cellular functions, including proliferation. The biological properties of CaMKII are regulated by multi-site phosphorylation and targeting via interactions with specific proteins. To investigate the role specific CaMKII phosphorylation sites play in controlling cell proliferation and cell cycle progression, we examined phosphorylation of CaMKII at two sites (T253 and T286) at various stages of the cell cycle, and also examined the effects of overexpression of wild-type (WT), T286D phosphomimic, T253D phosphomimic and T253V phosphonull forms of CaMKIIα in MDA-MB-231 breast cancer and SHSY5Y neuroblastoma cells on cellular proliferation and cell cycle progression. We demonstrate herein that whilst there is no change in total CaMKII expression or T286 phosphorylation throughout the cell cycle, a marked dephosphorylation of CaMKII at T253 occurs during the G₂ and/or M phases. Additionally, we show by molecular inhibition, as well as pharmacological activation, that protein phosphatase 2A (PP2A) is the phosphatase responsible for this dephosphorylation. Furthermore, we show that inducible overexpression of WT, T286D and T253V forms of CaMKIIα in MDA-MB-231 and SHSY5Y cells increases cellular proliferation, with no alteration in cell cycle profiles. By contrast, overexpression of a T253D phosphomimic form of CaMKIIα significantly decreases proliferation, and cells accumulate in mitosis, specifically in metaphase. Taken together, these results strongly suggest that the dephosphorylation of CaMKII at T253 is involved in controlling the cell cycle, specifically the metaphase–anaphase transition.     

Knockout of ATG5 leads to malignant cell transformation and resistance to Src family kinase inhibitor PP2

Autophagy can either promote or inhibit cell death in different cellular contexts. In this study, we investigated the role of autophagy in ATG5 knockout (KO) cell line established using CRISPR/Cas9 system. In ATG5 KO cells, RT‐PCR and immunoblot of LC3 confirmed the functional gene knockout. We found that knockout of ATG5 significantly increased proliferation of NIH 3T3 cells. In particular, autophagy deficiency enhanced susceptibility to cellular transformation as determined by an in vitro clonogenic survival assay and a soft agar colony formation assay. We also found that ATG5 KO cells had a greater migration ability as compared to wild‐type (WT) cells. Moreover, ATG5 KO cells were more resistant to treatment with a Src family tyrosine kinase inhibitor (PP2) than WT cells were. Cyto‐ID Green autophagy assay revealed that PP2 failed to induce autophagy in ATG5 KO cells. PP2 treatment decreased the percentage of cells in the S and G₂/M phases among WT cells but had no effect on cell cycle distribution of ATG5 KO cells, which showed a high percentage of cells in the S and G₂/M phases. Additionally, the proportion of apoptotic cells significantly decreased after treatment of ATG5 KO cells with PP2 in comparison with WT cells. We found that expression levels of p53 were much higher in ATG5 KO cells. The ATG5 KO seems to lead to compensatory upregulation of the p53 protein because of a decreased apoptosis rate. Taken together, our results suggest that autophagy deficiency can lead to malignant cell transformation and resistance to PP2.     

Inhibition of non-muscle myosin II leads to G0/G1 arrest of Wharton's jelly-derived mesenchymal stromal cells

Background aimsMesenchymal stromal cells (MSCs) have remarkable clinical potential for cell-based therapy. Wharton's jelly-derived mesenchymal stromal cells (WJ-MSCs) from umbilical cord share unique properties with both embryonic and adult stem cells. MSCs are found at low frequency in vivo, and their successful therapeutic application depends on rapid and efficient large-scale expansion in vitro. Non-muscle myosin II (NMII) has pivotal roles in different cellular activities, such as cell division, migration and differentiation. We performed this study to understand the role of NMII in proliferation and cell cycle progression in WJ-MSCs.MethodsWJ-MSCs were cultured in the presence of blebbistatin, and cell cycle analysis was performed using flow cytometry, proliferation kinetics, senescence assay and gene expression profile using polymerase chain reaction array.ResultsWhen cultured in the presence of blebbistatin, an inhibitor of NMII adenosine triphosphatase activity, WJ-MSCs exhibited dose-dependent reduction in proliferative potential along with increase in cell size and induction of early senescence. Inhibition of NMII activity also affected cell cycle progression in WJ-MSCs and led to an increase in the percentage of cells in G₀/G₁ phase with a corresponding reduction in the percentage of cells in G₂/M phase. Blebbistatin-induced G₀/G₁ arrest of WJ-MSCs was further associated with up-regulation of cell cycle inhibitory genes CDKN1A, CDKN2A and CDKN2B and down-regulation of numerous genes related to progression through S and M phases of the cell cycle.ConclusionsOur study demonstrates that inhibition of NMII activity in WJ-MSCs leads to G₀/G₁ arrest and alteration in the expression levels of certain key cell cycle-related genes.     

Cell cycle,cell size and mitochondrial activity of hybridoma cells during batch cultivation

Cell cycle, cell size and rhodamine 123 fluorescence in cell populations of two batch cultures were analysed and quantified with a fluorescence-activated cell sorter (FACS). Two cultures derived from either exponential or stationary phase innocula were investigated in order to demonstrate the dependency of the subsequent cell growth on innoculum condition. The results demonstrated that the level of activity of cells in the innoculum culture could have a significant effect on cellular activity during the initial phase of the inoculated culture, as it advances through its growth cycle. Positive correlation was found between the cell size and mitochondrial activity (as measured by rhodamine 123 uptake) with S and G₂ fractions as the cell progressed through the cell cycle. The enumeration of the fractions of cell cycle phases has helped in prediction of the changes in cell numbers following perturbation of the culture condition.     

Insulin-like growth factor II receptor-mediated intracellular retention of cathepsin B is essential for transformation of endothelial cells by Kaposi's sarcoma-associated herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV) is the pathological agent of Kaposi's sarcoma (KS), a tumor characterized by aberrant proliferation of endothelial-cell-derived spindle cells. Since in many cancers tumorigenesis is associated with an increase in the activity of the cathepsin family, we studied the role of cathepsins in KS using an in vitro model of KSHV-mediated endothelial cell transformation. Small-molecule inhibitors and small interfering RNA (siRNA) targeting CTSB, but not other cathepsins, inhibited KSHV-induced postconfluent proliferation and the formation of spindle cells and foci of dermal microvascular endothelial cells. Interestingly, neither CTSB mRNA nor CTSB protein levels were induced in endothelial cells latently infected with KSHV. Secretion of CTSB was strongly diminished upon KSHV infection. Increased targeting of CTSB to endosomes was caused by the induction by KSHV of the expression of insulin-like growth factor-II receptor (IGF-IIR), a mannose-6-phosphate receptor (M6PR) that binds to cathepsins. Inhibition of IGF-IIR/M6PR expression by siRNA released CTSB for secretion. In contrast to the increased cathepsin secretion observed in most other tumors, viral inhibition of CTSB secretion via induction of an M6PR is crucial for the transformation of endothelial cells.     

敲低肿瘤细胞来源的颗粒体蛋白前体抑制C57BL/6J小鼠黑色素移植瘤的生长

颗粒体蛋白前体 (progranulin, PGRN)在多种肿瘤中过表达。但PGRN在黑色素瘤发生发展中的作用尚无报道。为探究PGRN在黑色素肿瘤中的作用,本研究采用CRISPR-Cas9基因编辑技术建立了稳定敲低PGRN的小鼠黑色素瘤B16细胞株B16-PGRNlow。MTS法和BrdU掺入结合流式细胞（计量）术分析证明,敲低PGRN不影响B16细胞的细胞周期和增殖。将B16-ctrl（对照）和B16-PGRNlow细胞分别皮下接种野生型（WT）和PGRN敲除（KO）的C57BL/6J小鼠,比较观察黑色素移植瘤体积大小。移植瘤形成20 d后,与B16-ctrl细胞接种的移植瘤比较,无论在WT还是在KO荷瘤小鼠,B16-PGRNlow形成的移植瘤体积明显减小（WT鼠：P<0.05;KO鼠：P<0.01）。然而,比较B16-PGRNlow或B16-ctrl在WT鼠与KO鼠形成的移植瘤体积大小,并无显著差异,提示B16肿瘤细胞PGRN而非宿主PGRN影响移植瘤的生长。流式细胞术分析显示,在荷B16-PGRNlow移植瘤的WT型小鼠脾和淋巴结中,CD4+、CD8+T细胞数（百分比）比荷B16-ctrl移植瘤的WT鼠脾和淋巴结的CD4+、CD8+T细胞数明显增多（P<0.05,P<0.01）,而在KO鼠却未见明显差异。上述结果证明,敲低肿瘤细胞PGRN可抑制黑色素移植瘤的生长。上述结果还提示,抑制PGRN在黑色瘤的表达可引起脾和淋巴结CD4+和CD8+T细胞增加,提高宿主的细胞免疫能力。其机制尚待进一步研究。本文的发现为PGRN作为黑色素瘤治疗的潜在靶点提供了新证据。     

Kaposi's Sarcoma-Associated Herpesvirus Viral Interferon Regulatory Factor 4 (vIRF4) Perturbs the G1-S Cell Cycle Progression via Deregulation of the cyclin D1 Gene

Kaposi''s sarcoma-associated herpesvirus (KSHV) infection modulates the host cell cycle to create an environment optimal for its viral-DNA replication during the lytic life cycle. We report here that KSHV vIRF4 targets the β-catenin/CBP cofactor and blocks its occupancy on the cyclin D1 promoter, suppressing the G₁-S cell cycle progression and enhancing KSHV replication. This shows that KSHV vIRF4 suppresses host G₁-S transition, possibly providing an intracellular milieu favorable for its replication.     

Gravitational force modulates G2/M phase exit in mechanically unloaded myoblasts

Prolonged spaceflight gives rise to muscle loss and reduced strength, a condition commonly referred to as space atrophy. During exposure to microgravity, skeletal muscle myoblasts are mechanically unloaded and respond with attenuated cell proliferation, slowed cell cycle progression, and modified protein expression. To elucidate the underlying mechanisms by which muscle mass declines in response to prolonged microgravity exposure, we grew C2C12 mouse muscle cells under conditions of simulated microgravity (SM) and analyzed their proliferative capacity, cell cycle progression, and cyclin B and D expression. We demonstrated that the retarded cell growth observed in SM was correlated with an approximate 16 h delay in G₂/M phase progression, where cells accumulated specifically between the G₂ checkpoint and the onset of anaphase, concomitantly with a positive expression for cyclin B. The effect was specific for gravitational mechanical unloading as cells grown under conditions of hypergravity (HG, 4 g) for similar durations of time exhibited normal proliferation and normal cell cycle progression. Our results show that SM and HG exert phenomenological distinct responses over cell cycle progression. The deficits of SM can be restored by terrestrial gravitational force, whereas the effects of HG are indistinguishable from the 1 g control. This suggests that the mechanotransduction apparatus of cells responds differently to mechanical unloading and loading.     

Overexpression of a novel osteopetrosis-related gene CCDC154 suppresses cell proliferation by inducing G2/M arrest

Osteopetrosis, a disorder of skeletal bone, can cause death during childhood. We previously described a new spontaneous autosomal recessive osteopetrosis mouse mutant, “new toothless” (ntl). In this study, we reported for the first time the identification, cloning and characterization of the coiled-coil domain-containing 154 (CCDC154), a novel gene whose deletion of ~5 kb sequence including exons 1–6 was completely linked to the ntl mutant. The CCDC154 was conserved between mouse and human and is wildly expressed in mouse tissues. The cellular localization of CCDC154 was in the early endosomes. Overexpression of CCDC154 inhibited cell proliferation of HEK293 cells by inducing G₂/M arrest. CCDC154 also inhibited tumor cell growth, and the soft agar assay revealed a significant decrease of the colony size of Hela cells upon transfection of CCDC154. Our results indicate that CCDC154 is a novel osteopetrosis-related gene involved in cell cycle regulation and tumor suppression growth.     

Overexpression of a novel osteopetrosis-related gene CCDC154 suppresses cell proliferation by inducing G2/M arrest

Osteopetrosis, a disorder of skeletal bone, can cause death during childhood. We previously described a new spontaneous autosomal recessive osteopetrosis mouse mutant, “new toothless” (ntl). In this study, we reported for the first time the identification, cloning and characterization of the coiled-coil domain-containing 154 (CCDC154), a novel gene whose deletion of ~5 kb sequence including exons 1–6 was completely linked to the ntl mutant. The CCDC154 was conserved between mouse and human and is wildly expressed in mouse tissues. The cellular localization of CCDC154 was in the early endosomes. Overexpression of CCDC154 inhibited cell proliferation of HEK293 cells by inducing G₂/M arrest. CCDC154 also inhibited tumor cell growth, and the soft agar assay revealed a significant decrease of the colony size of Hela cells upon transfection of CCDC154. Our results indicate that CCDC154 is a novel osteopetrosis-related gene involved in cell cycle regulation and tumor suppression growth.     

Normal versus abnormal cell proliferation

The most recent findings on the molecular and cellular characterization of normal and abnormal cell proliferation are summarized. They include molecular spectroscopy, nucleic acid conformation, protein modifications, premature chromosome condensation, nuceoli changes, nuclear and cell morphometry, image analysis, flow microfluorimetry, and time-lapse cinematography. Biophysical and biochemical evidence in favor or against two cycles of chromatin condensation, followed by two abrupt random decondensations, per cell cycle are presented. Other biphasic changes at the molecular and cellular levels that favor the existence of two random transitions, or restriction points, per cell cycle are discussed. A comprehensive unitary model of the cell cycle is then outlined; this model is able to explain most findings on continuously dividing cells and on quiescent cells induced to proliferate. Within this analytical framework the physical-chemical and biological properties are given, in either normal or tumor cells, for the various types of “noncycling” cells that are here viewed as necessary steps in mammalian cell growth rather than separate states. The implications of the coupling of higher-order chromatin structure with cell geometry and growth, high in fibroblast-like cells but low in transformed cells, are also discussed. Molecular mechanisms likely responsible for the chromatin conformational changes occurring at the G₀→G₁, G₁→S, G₂→M transitions are finally discussed in terms of polyelectrolyte theory.