Development of a novel, high-efficacy oncolytic herpes simplex virus type 1 platform equipped with two distinct retargeting modalities.

To retarget oncolytic herpes simplex virus (oHSV) to cancer-specific antigens, we designed a novel, double-retargeted oHSV platform that uses single-chain antibodies (scFvs) incorporated into both glycoprotein H and a bispecific adapter expressed from the viral genome to mediate infection predominantly via tumor-associated antigens. Successful retargeting was achieved using a nectin-1-detargeted HSV that remains capable of interacting with herpesvirus entry mediator (HVEM), the second canonical HSV entry receptor, and is, therefore, recognized by the adapter consisting of the virus-binding N-terminal 82 residues of HVEM fused to the target-specific scFv. We tested both an epithelial cell adhesion molecule (EpCAM)- and a human epidermal growth factor receptor 2-specific scFv separately and together to target cells expressing one, the other, or both receptors. Our results show not only dose-dependent, target receptor-specific infection in vitro, but also enhanced virus spread compared with single-retargeted virus. In addition, we observed effective infection and spreading of the EpCAM double-retargeted virus in vivo. Remarkably, a single intravenous dose of the EpCAM-specific virus eliminated all detectable tumors in a subcutaneous xenograft model, and the same intravenous dose seemed to be harmless in immunocompetent FVB/N mice. Our findings suggest that our double-retargeted oHSV platform can provide a potent, versatile, and systemically deliverable class of anti-cancer therapeutics that specifically target cancer cells while ensuring safety.

NADH elevation during chronic hypoxia leads to VHL-mediated HIF-1α degradation via SIRT1 inhibition.

BACKGROUND: Under conditions of hypoxia, cancer cells with hypoxia inducible factor-1α (HIF-1α) from heterogeneous tumor cells show greater aggression and progression in an effort to compensate for harsh environmental conditions. Extensive study on the stability of HIF-1α under conditions of acute hypoxia in cancer progression has been conducted, however, understanding of its involvement during the chronic phase is limited. METHODS: In this study, we investigated the effect of SIRT1 on HIF1 stability in a typical chronic hypoxic conditon that maintains cells for 24 h under hypoxia using Western blotting, co-IP, measurement of intracellular NAD + and NADH levels, semi-quantitative RT-PCR analysis, invasion assay, gene knockdown. RESULTS: Here we demonstrated that the high concentration of pyruvate in the medium, which can be easily overlooked, has an effect on the stability of HIF-1α. We also demonstrated that NADH functions as a signal for conveyance of HIF-1α degradation via the SIRT1 and VHL signaling pathway under conditions of chronic hypoxia, which in turn leads to attenuation of hypoxically strengthened invasion and angiogenic activities. A steep increase in the level of NADH occurs during chronic hypoxia, leading to upregulation of acetylation and degradation of HIF-1α via inactivation of SIRT1. Of particular interest, p300-mediated acetylation at lysine 709 of HIF-1α is recogonized by VHL, which leads to degradation of HIF-1α via ubiquitin/proteasome machinary under conditions of chronic hypoxia. In addition, we demonstrated that NADH-elevation-induced acetylation and subsequent degradation of HIF-1α was independent of proline hydroxylation. CONCLUSIONS: Our findings suggest a critical role of SIRT1 as a metabolic sensor in coordination of hypoxic status via regulation of HIF-1α stability. These results also demonstrate the involvement of VHL in degradation of HIF-1α through recognition of PHD-mediated hydroxylation in normoxia and p300-mediated HIF-1α acetylation in hypoxia.

Inhibition of HIF-1α by Atorvastatin During 131I-RTX Therapy in Burkitt's Lymphoma Model.

BACKGROUNDS: Radioimmunotherapy (RIT) serves as a targeted therapy for non-Hodgkin lymphomas (NHL). Although HIF(Hypoxia-inducible factors)-1α is an important biomarker during radiation therapy, its role in NHL is unclear. Atorvastatin (ATV) is used as a combination drug for chemotherapy. METHODS: We investigated whether ATV downregulated tumor radio-resistance and enhanced the anticancer effect of 131I-RTX (rituximab) in Raji xenograft mouse models. First, the increased uptake and enhanced therapeutic effect of 131I-RTX by ATV was confirmed using molecular imaging in Raji xenograft subcutaneous model and orthotropic model with SPECT and IVIS images. Second, we examined the profile of differentially expressed miRNAs using miRNA array. RESULTS: We found that miR-346 inhibited HIF-1α/VEGF (Vascular endothelial growth factor) during ATV combination therapy with 131I-RTX. The underlying mechanism of ATV involved induction of anti-angiogenesis and radiosensitivity by downregulating HIF-1α in Raji cells. CONCLUSION: Our findings suggested that combination therapy with ATV and 131I-RTX is a promising strategy for enhancing the potency of 131I-RTX therapy in poorly responding patients and those with radio-resistance.

Cluh plays a pivotal role during adipogenesis by regulating the activity of mitochondria.

Cluh is a cytosolic protein that is known to specifically bind the mRNAs of nuclear-encoded mitochondrial proteins and play critical roles in mitochondrial biogenesis. Here, we report the role of Cluh in adipogenesis. Our study shows that mRNA expression of Cluh is stimulated during adipogenesis, and that cAMP/Creb signalling increases its transcription. Cluh depletion impaired proper adipocyte differentiation, with reductions seen in lipid droplets and adipogenic marker gene expression. Interestingly, the inductions of the brown adipocyte-specific genes, Ucp1, Cidea and Cox7a1, are severely blocked by Cluh depletion during brown adipogenesis. Mitochondrial respiration and the stability of mRNAs encoding mitochondrial proteins are reduced by Cluh depletion during brown adipogenesis. These results suggest that Cluh, which is induced during adipogenesis, promotes the post-transcriptional regulation of mitochondrial proteins and supports differentiation.

Protein arginine methyltransferase 5 is implicated in the aggressiveness of human hepatocellular carcinoma and controls the invasive activity of cancer cells.

Protein arginine methyltransferase 5 (PRMT5) is a protein that catalyzes transfer of methyl groups to the arginine residues of proteins and is involved in diverse cellular and biological responses. While the participation of PRMT5 in cancer progression has been increasingly documented, its association with the invasive phenotype currently remains poorly understood. In the present study, we revealed that PRMT5 is overexpressed in human hepatocellular carcinoma (HCC) and in colon cancer and its depletion leads to the suppression of cell invasive activity via the reduction of the expression of MMP-2. Real-time quantitative RT-PCR analysis of 120 HCC patient tissues revealed the overexpression of PRMT5 in HCC and the association of PRMT5 with aggressive clinicopathological parameters, such as poorer differentiation (P=0.004), more frequent hepatic vein invasion (P=0.019), larger tumor size (P=0.011) and higher α-fetoprotein levels (P=0.020). Similarly to the data obtained with HCC, overexpression of PRMT5 was also displayed in colon cancer tissues, compared to matched non-tumor regions. Consistent with the significant association of the overexpression of PRMT5 with hepatic vein invasion in patient specimens, PRMT5 depletion via siRNA transfection led to a marked reduction in the invasion rate in both HCC and colon cancer cells. Reduced invasion associated with PRMT5 depletion was accompanied by a decrease in the expression of MMP-2. Collectively, our results indicated that PRMT5 overexpression in HCC and colon cancer cells contributed to their acquisition of aggressive characteristics, such as invasiveness, thus presenting a promising therapeutic target for the treatment of these diseases.

Mael is essential for cancer cell survival and tumorigenesis through protection of genetic integrity.

Germ line-specific genes are activated in somatic cells during tumorigenesis, and are accordingly referred to as cancer germline genes. Such genes that act on piRNA (Piwi-interacting RNA) processing play an important role in the progression of cancer cells. Here, we show that the spermatogenic transposon silencer maelstrom (Mael), a piRNA-processing factor, is required for malignant transformation and survival of cancer cells. A specific Mael isoform was distinctively overexpressed in diverse human cancer cell lines and its depletion resulted in cancer-specific cell death, characterized by apoptosis and senescence, accompanied by an increase in reactive oxygen-species and DNA damage. These biochemical changes and death phenotypes induced by Mael depletion were dependent on ATM. Interestingly Mael was essential for Myc/Ras-induced transformation, and its overexpression inhibited Ras-induced senescence. In addition, Mael repressed retrotransposon activity in cancer cells. These results suggest that Mael depletion induces ATM-dependent DNA damage, consequently leading to cell death specifically in cancer cells. Moreover, Mael possesses oncogenic potential that can protect against genetic instability.

p31comet-Induced Cell Death Is Mediated by Binding and Inactivation of Mad2.

Mad2, a key component of the spindle checkpoint, is closely associated with chromosomal instability and poor prognosis in cancer. p31comet is a Mad2-interacting protein that serves as a spindle checkpoint silencer at mitosis. In this study, we showed that p31comet-induced apoptosis and senescence occur via counteraction of Mad2 activity. Upon retroviral transduction of p31comet, the majority of human cancer cell lines tested lost the ability to form colonies in a low-density seeding assay. Cancer cells with p31comet overexpression underwent distinct apoptosis and/or senescence, irrespective of p53 status, confirming the cytotoxicity of p31comet. Interestingly, both cytotoxic and Mad2 binding activities were eliminated upon deletion of the C-terminal 30 amino acids of p31comet. Point mutation or deletion of the region affecting Mad2 binding additionally abolished cytotoxic activity. Consistently, wild-type Mad2 interacting with p31comet, but not its non-binding mutant, inhibited cell death, indicating that the mechanism of p31comet-induced cell death involves Mad2 inactivation. Our results clearly suggest that the regions of p31comet affecting interactions with Mad2, including the C-terminus, are essential for induction of cell death. The finding that p31comet-induced cell death is mediated by interactions with Mad2 that lead to its inactivation is potentially applicable in anticancer therapy.

SIRT1 deacetylates and stabilizes hypoxia-inducible factor-1α (HIF-1α) via direct interactions during hypoxia.

Upon shift to a hypoxic environment, cellular HIF-1α protein is stabilized, with a rapid decline in oxygen-sensitive hydroxylation. Several additional post-translational modifications of HIF-1α are critical in controlling protein stability during hypoxia. In the present study, we showed that SIRT1 stabilizes HIF-1α via direct binding and deacetylation during hypoxia. SIRT1 depletion or inactivation led to reduced hypoxic HIF-1α accumulation, accompanied by an increase in HIF-1α acetylation. Impaired HIF-1α accumulation was recovered upon inhibition of 26S proteasome activity, indicating that SIRT1 is essential for HIF-1α stabilization during hypoxia. Consistently, HIF-1α accumulation was enhanced upon overexpression of wild-type SIRT1, but not its dominant-negative form. SIRT1-mediated accumulation of HIF-1α protein led to increased expression of HIF-1α target genes, including VEGF, GLUT1 and MMP2, and ultimate promotion of cancer cell invasion. These findings collectively imply that hypoxic HIF-1α stabilization requires SIRT1 activation. Furthermore, SIRT1 protection of HIF-1α from acetylation may be a prerequisite for stabilization and consequent enhancement of cell invasion.

RRM1 maintains centrosomal integrity via CHK1 and CDK1 signaling during replication stress.

DNA lesion-induced centrosomal abnormalities during the replication phase are relatively unknown. Here, we report that RNAi-mediated depletion of RRM1 induces cell-cycle arrest at the replication phase, along with severe DNA damage and centrosomal amplification. Interestingly, CHK1 depletion synergistically increased RRM1-depletion-induced centrosomal amplification. In response to hydroxyurea, CHK1 was delocalized from the centrosome by RRM1 depletion. Moreover, CDK1, which functions in centrosome separation and is inhibited by CHK1, was found to be essential for RRMI1-depletion-induced centrosomal amplification. Thus, we herein demonstrate that RRM1 preserves chromosomal stability via the CHK1- and CDK1-dependent stabilization of the centrosomal integrity at the replication stage.

Inhibition of HAS2 induction enhances the radiosensitivity of cancer cells via persistent DNA damage.

Hyaluronan synthase 2 (HAS2), a synthetic enzyme for hyaluronan, regulates various aspects of cancer progression, including migration, invasion and angiogenesis. However, the possible association of HAS2 with the response of cancer cells to anticancer radiotherapy, has not yet been elucidated. Here, we show that HAS2 knockdown potentiates irradiation-induced DNA damage and apoptosis in cancer cells. Upon exposure to radiation, all of the tested human cancer cell lines exhibited marked (up to 10-fold) up-regulation of HAS2 within 24h. Inhibition of HAS2 induction significantly reduced the survival of irradiated radioresistant and -sensitive cells. Interestingly, HAS2 depletion rendered the cells to sustain irradiation-induced DNA damage, thereby leading to an increase of apoptotic death. These findings indicate that HAS2 knockdown sensitizes cancer cells to radiation via persistent DNA damage, further suggesting that the irradiation-induced up-regulation of HAS2 contributes to the radioresistance of cancer cells. Thus, HAS2 could potentially be targeted for therapeutic interventions aimed at radiosensitizing cancer cells.

SIRT1 suppresses cellular accumulation of ß-TrCP E3 ligase via protein degradation.

ß-Transducin repeat-containing protein (ß-TrCP), an E3 ligase, promotes the degradation of substrate proteins in response to various stimuli. Even though several ß-TrCP substrates have been identified to date, limited information of its upstream regulators is available. Here, we showed that SIRT1 suppresses ß-TrCP protein synthesis via post-translational degradation. SIRT1 depletion led to a significant increase in the ß-TrCP accumulation without affecting the mRNA level. Consistently, ß-TrCP protein accumulation induced by resveratrol was further enhanced upon SIRT1 depletion. Rescue of SIRT1 reversed the effect of resveratrol, leading to reduced ß-TrCP protein levels. Proteasomal inhibition led to recovery of ß-TrCP in cells with SIRT1 overexpression. Notably, the recovered ß-TrCP colocalized mostly with SIRT1. Thus, SIRT1 acts as a negative regulator of ß-TrCP synthesis via promoting protein degradation.

SIRT1 suppresses activating transcription factor 4 (ATF4) expression in response to proteasome inhibition.

The synthetic machinery of ATF4 (activating transcription factor 4) is activated in response to various stress conditions involved in nutrient restriction, endoplasmic reticulum homeostasis, and oxidation. Stress-induced inhibition of proteasome activity triggers the unfolded protein response and endoplasmic reticulum stress, where ATF4 is crucial for consequent biological events. In the current study, we showed that the NAD(+)-dependent deacetylase, SIRT1, suppresses ATF4 synthesis during proteasome inhibition. SIRT1 depletion via transfection of specific siRNA into HeLa cells resulted in a significant increase in ATF4 protein, which was observed specifically in the presence of the proteasome inhibitor MG132. Consistent with SIRT1 depletion data, transient transfection of cells with SIRT1-overexpressing plasmid induced a decrease in the ATF4 protein level in the presence of MG132. Interestingly, however, ATF4 mRNA was not affected by SIRT1, even in the presence of MG132, indicating that SIRT1-induced suppression of ATF4 synthesis occurs under post-transcriptional control. Accordingly, we propose that SIRT1 serves as a negative regulator of ATF4 protein synthesis at the post-transcriptional level, which is observed during stress conditions, such as proteasome inhibition.

SIRT1 interacts with and protects glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from nuclear translocation: implications for cell survival after irradiation.

Upon apoptotic stimulation, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a cytosolic enzyme normally active in glycolysis, translocates into the nucleus and activates an apoptotic cascade therein. In the present work, we show that SIRT1 prevents nuclear translocation of GAPDH via interaction with GAPDH. SIRT1 depletion triggered nuclear translocation of cytosolic GAPDH even in the absence of apoptotic stress. Such translocation was not, however, observed when SIRT1 enzymatic activity was inhibited, indicating that SIRT1 protein per se, rather than the deacetylase activity of the protein, is required to inhibit GAPDH translocation. Upon irradiation, SIRT1 prevented irradiation-induced nuclear translocation of GAPDH, accompanied by interaction of SIRT1 and GAPDH. Thus, SIRT1 functions to retain GAPDH in the cytosol, protecting the enzyme from nuclear translocation via interaction with these two proteins. This serves as a mechanism whereby SIRT1 regulates cell survival upon induction of apoptotic stress by means that include irradiation.

Mitomycin C and doxorubicin elicit conflicting signals by causing accumulation of cyclin E prior to p21WAF1/CIP1 elevation in human hepatocellular carcinoma cells.

Proteins involved in the G1 phase of the cell cycle are aberrantly expressed, sometimes in mutated forms, in human cancers including human hepatocellular carcinoma. Upon attack by a DNA-damaging anticancer drug, a cell arrests at the G1 phase; this is a safety feature prohibiting entry of DNA-damaged cells into S-phase. p21WAF1/CIP1 prevents damaged cells from progressing to the next cell cycle. Here, we show that, in response to mitomycin C and doxorubicin, human hepatocellular carcinoma cells generate conflicting signals, mediated by cyclin E and p21WAF1/CIP1, which respectively accelerates and represses cell cycle transition. Exposure to these anticancer drugs led to rapid accumulation of cyclin E in both p53-proficient HepG2 and p53-deficient Hep3B cells. Such anticancer drug-induced cyclin E accumulation influenced the G1-S-phase transition, but not DNA fragmentation-mediated death. In p53-proficient HepG2 cells, accumulation of cyclin E was followed by an increase in the level of p53-dependent p21WAF1/CIP1, thereby inhibiting further the G1-S-phase transition. Sublethal drug concentrations also induced rapid accumulation of cyclin E, but p21WAF1/CIP1 accumulation was delayed, further facilitating the G1-S-phase transition. Eventually, most cells arrested in G2/M. Thus, mitomycin C- or doxorubicin-induced conflicting signals, mediated by cyclin E and p21WAF1/CIP1, are in play in human hepatocellular carcinoma cells. Damaged G1 cells either immediately enter S-phase, or do not do so at all, depending on the extent of DNA damage.

Cells with dysfunctional telomeres are susceptible to reactive oxygen species hydrogen peroxide via generation of multichromosomal fusions and chromosomal fragments bearing telomeres.

During genotoxic stress, reactive oxygen species hydrogen peroxide (H(2)O(2)) is a prime mediator of the DNA damage response. Telomeres function both to assist in DNA damage repair and to inhibit chromosomal end-to-end fusion. Here, we show that telomere dysfunction renders cells susceptible to H(2)O(2), via generation of multichromosomal fusion and chromosomal fragments. H(2)O(2) caused formation of multichromosomal end-to-end fusions involving more than three chromosomes, preferentially when telomeres were erosive. Interestingly, extensive chromosomal fragmentation (yielding small-sized fragments) occurred only in cells exhibiting such multichromosomal fusions. Telomeres were absent from fusion points, being rather present in the small fragments, indicating that H(2)O(2) cleaves chromosomal regions adjacent to telomeres. Restoration of telomere function or addition of the antioxidant N-acetylcysteine prevented development of chromosomal aberrations and rescued the observed hypersensitivity to H(2)O(2). Thus, chromosomal regions adjacent to telomeres become sensitive to reactive oxygen species hydrogen peroxide when telomeres are dysfunctional, and are cleaved to produce multichromosomal fusions and small chromosomal fragments bearing the telomeres.

Paclitaxel stimulates chromosomal fusion and instability in cells with dysfunctional telomeres: implication in multinucleation and chemosensitization.

The anticancer effect of paclitaxel is attributable principally to irreversible promotion of microtubule stabilization and is hampered upon development of chemoresistance by tumor cells. Telomere shortening, and eventual telomere erosion, evoke chromosomal instability, resulting in particular cellular responses. Using telomerase-deficient cells derived from mTREC-/-p53-/- mice, here we show that, upon telomere erosion, paclitaxel propagates chromosomal instability by stimulating chromosomal end-to-end fusions and delaying the development of multinucleation. The end-to-end fusions involve both the p- and q-arms in cells in which telomeres are dysfunctional. Paclitaxel-induced chromosomal fusions were accompanied by prolonged G2/M cell cycle arrest, delayed multinucleation, and apoptosis. Telomere dysfunctional cells with mutlinucleation eventually underwent apoptosis. Thus, as telomere erosion proceeds, paclitaxel stimulates chromosomal fusion and instability, and both apoptosis and chemosensitization eventually develop.

Establishment of a reliable dual-vector system for the phage display of antibody fragments.

To resolve some of the technical limitations in a phage-displayed Fab library, we have designed two dual-vector systems, DVS-I and DVS-II, composed of a set of replicon-compatible plasmid (pLA-1 or pLT-2) for producing soluble L chain fragments and phagemid (pHf1g3T-1 or pHf1g3A-2) for expressing Fd (V(H)+C(H1))-DeltapIII fusion molecules as well as a genotype-phenotype linkage. Compared to the DVS-I (pLA-1 and pHf1g3T-1), the DVS-II (pLT-2 and pHf1g3A-2) showed stable transformation efficiency regardless of the order of the vectors introduced into the host cells. In addition, expression of soluble Fab molecules with antigen-binding reactivity, recombinant phage titer and display level of functional Fab-DeltapIII on the phage progenies of the DVS-II were comparable with a conventional phage display system using a single phagemid vector. More importantly, the phage displaying target-specific Fab-DeltapIII molecules was successfully enriched by panning, which allows isolation of the pHf1g3A-2 phagemid encoding antigen-specific Fd molecules. We believe that the DVS-II may provide a valuable tool in the construction of a combinatorial phage-displayed Fab library with large diversity. Furthermore, it can be readily applied to isolation of desired antibody clones if L chain promiscuity of antibodies in determining antigen-binding specificity is considered, or in guided-selection or chain shuffling of mAbs of non-human origin.

Enhancing phage display of antibody fragments using gIII-amber suppression.

The effect of utilizing Ex12 helper phage, a mutant M13K07 helper having two amber codons at the gIII (gIII-amber), in combination with Escherichia coli host strains belonging to the supE genotype on improving the phage display of antibody fragments was investigated. Because of an inefficient read-through of the UAG codons, Ex12 helper phage produced approximately 10% of the intracellular wt pIII in the supE host cells compared to M13K07. The phage progenies rescued from the supE XL-1 Blue MRF' strain carrying the recombinant phagemid, pCMTG-SP112, by Ex12 helper phage displayed both antibody-DeltapIII fusion and wt pIII at a ratio of 1:1.5, and achieved a 50-fold greater display of the antibody-DeltapIII compared to those obtained by a conventional phage rescue using M13K07. Additionally observed were a 100-fold increase in antigen-binding functionality and a drastic improvement on antigen-specific panning efficiency by the phage progenies. Our approach permits the display of at least one antibody fragment as well as more than one copy of wt pIII on the surface of recombinant phages, and this would make the phagemid-based phage display technology more practical and reliable.