Enhanced tumor targeting and timely viral release of mesenchymal stem cells/oncolytic virus complex due to GRP78 and inducible E1B55K expressions greatly increase the antitumor effect of systemic treatment.

Systemic delivery of oncolytic viruses has been widely regarded as an impractical option for antitumor treatment. Here, we selected two target genes as leading components, and significant therapeutic effects were obtained by simultaneously reducing the expression of transforming growth factor ß 1 (TGF-ß1) and heat shock protein 27 (HSP27) in various cancer cell types. Downregulation of HSP27 reduced the cellular levels of tumor progression-related proteins, and the simultaneous downregulation of HSP27 and TGF-ß1 increased tumor cell death beyond that observed with TGF-ß1 downregulation alone. To increase the potential for systemic administration, we generated modified mesenchymal stem cells (MSCs) to act as oncolytic adenovirus factories and carriers and assessed bioavailability in tumors after MSC injection. The MSCs were modified to express 78-kDa glucose-regulated protein (GRP78) and adenovirus early-region 1B 55 kDa (E1B55K). The tightly controlled inducible system permitted selective timing of viral release from carrier MSCs within the tumor. This approach significantly improved viral production, tumor targeting, timely viral release at the tumor site, and antitumor efficacy of the oncolytic adenovirus. These combined results demonstrate that engineered MSCs can significantly enhance the antitumor effects of oncolytic viruses without adverse safety issues, which may greatly extend the clinical applicability of oncolytic adenoviruses.

Enhanced oncolytic adenoviral production by downregulation of death-domain associated protein and overexpression of precursor terminal protein.

Adequate viral replication in tumor cells is the key to improving the anti-cancer effects of oncolytic adenovirus therapy. In this study, we introduced short hairpin RNAs against death-domain associated protein (Daxx), a repressor of adenoviral replication, and precursor terminal protein (pTP), an initiator of adenoviral genome replication, into adenoviral constructs to determine their contributions to viral replication. Both Daxx downregulation and pTP overexpression increased viral production in variety of human cancer cell lines, and the enhanced production of virus progeny resulted in more cell lysis in vitro, and tumor regression in vivo. We confirmed that increased virus production by Daxx silencing, or pTP overexpression, occurred using different mechanisms by analyzing levels of adenoviral protein expression and virus production. Specifically, Daxx downregulation promoted both virus replication and oncolysis in a consecutive manner by optimizing IVa2-based packaging efficiency, while pTP overexpression by increasing both infectious and total virus particles but their contribution to increased viral production may have been damaged to some extent by their another contribution to apoptosis and autophagy. Therefore, introducing both Daxx shRNA and pTP in virotherapy may be a suitable strategy to increase apoptotic tumor-cell death and to overcome poor viral replication, leading to meaningful reductions in tumor growth in vivo.

High levels of Daxx due to low cellular levels of HSP25 in murine cancer cells result in inefficient adenovirus replication.

When the adenoviral protein E1B55K binds death domain-associated protein (Daxx), the proteasome-dependent degradation of Daxx is initiated, and adenoviral replication is effectively maintained. Here, we show that the cellular levels of Daxx differ between human and mouse cancer cell lines. Specifically, we observed higher cellular Daxx levels and the diminished replication of oncolytic adenovirus in mouse cancer cell lines, suggesting that cellular Daxx levels limit the replication of oncolytic adenoviruses that lack E1B55K in murine cells. Indeed, the replication of oncolytic adenoviruses that lack E1B55K was significantly increased following infection with oncolytic adenovirus expressing Daxx-specific shRNA. Cellular Daxx levels were decreased in mouse cells expressing heat shock protein 25 (HSP25; homolog of human HSP27) following heat shock or stable transfection with HSP25-bearing plasmids. Furthermore, Daxx expression in murine cell lines was primarily regulated at the transcriptional level via HSP25-mediated inhibition of the nuclear translocation of the signal transducer and activator of transcription 3 (stat3) protein, which typically upregulates Daxx transcription. Conversely, human HSP27 enhanced stat3 activity to increase Daxx transcription. Interestingly, human Daxx, but not mouse Daxx, was degraded as normal by ubiquitin-dependent lysosomal degradation; however, HSP27 downregulation induced the ubiquitin-independent proteasomal degradation of Daxx.

TGF-ß downregulation-induced cancer cell death is finely regulated by the SAPK signaling cascade.

Transforming growth factor (TGF)-ß signaling is increasingly recognized as a key driver in cancer. In progressive cancer tissues, TGF-ß promotes tumor formation, and its increased expression often correlates with cancer malignancy. In this study, we utilized adenoviruses expressing short hairpin RNAs against TGF-ß1 and TGF-ß2 to investigate the role of TGF-ß downregulation in cancer cell death. We found that the downregulation of TGF-ß increased the phosphorylation of several SAPKs, such as p38 and JNK. Moreover, reactive oxygen species (ROS) production was also increased by TGF-ß downregulation, which triggered Akt inactivation and NOX4 increase-derived ROS in a cancer cell-type-specific manner. We also revealed the possibility of substantial gene fluctuation in response to TGF-ß downregulation related to SAPKs. The expression levels of Trx and GSTM1, which encode inhibitory proteins that bind to ASK1, were reduced, likely a result of the altered translocation of Smad complex proteins rather than from ROS production. Instead, both ROS and ROS-mediated ER stress were responsible for the decrease in interactions between ASK1 and Trx or GSTM1. Through these pathways, ASK1 was activated and induced cytotoxic tumor cell death via p38/JNK activation and (or) induction of ER stress.

Targeting mutant KRAS with CRISPR-Cas9 controls tumor growth.

KRAS is the most frequently mutated oncogene in human tumors, and its activating mutations represent important therapeutic targets. The combination of Cas9 and guide RNA from the CRISPR-Cas system recognizes a specific DNA sequence and makes a double-strand break, which enables editing of the relevant genes. Here, we harnessed CRISPR to specifically target mutant KRAS alleles in cancer cells. We screened guide RNAs using a reporter system and validated them in cancer cells after lentiviral delivery of Cas9 and guide RNA. The survival, proliferation, and tumorigenicity of cancer cells in vitro and the growth of tumors in vivo were determined after delivery of Cas9 and guide RNA. We identified guide RNAs that efficiently target mutant KRAS without significant alterations of the wild-type allele. Doxycycline-inducible expression of this guide RNA in KRAS-mutant cancer cells transduced with a lentiviral vector encoding Cas9 disrupted the mutant KRAS gene, leading to inhibition of cancer cell proliferation both in vitro and in vivo. Intra-tumoral injection of lentivirus and adeno-associated virus expressing Cas9 and sgRNA suppressed tumor growth in vivo, albeit incompletely, in immunodeficient mice. Expression of Cas9 and the guide RNA in cells containing wild-type KRAS did not alter cell survival or proliferation either in vitro and in vivo. Our study provides a proof-of-concept that CRISPR can be utilized to target driver mutations of cancers in vitro and in vivo.

Down-Regulation of TGF-ß Expression Sensitizes the Resistance of Hepatocellular Carcinoma Cells to Sorafenib.

PURPOSE: Sorafenib, a multikinase inhibitor, is the standard therapy for patients with advanced-stage hepatocellular carcinoma (HCC). However, resistance develops to the treatment, therefore, we tried to unravel the underlying mechanism in the resistance of HCC cells to sorafenib via the development of more effective therapeutic strategies. MATERIALS AND METHODS: Various liver cancer cell lines were treated with either sorafenib only or with sorafenib after infection of adenovirus expressing short hairpin RNA (shRNA) against transforming growth factor-ß (TGF-ß) and p38 activity was examined using western blotting. RESULTS: p38 MAP kinase activity was inhibited by low concentrations of sorafenib, which could potentially lead to sorafenib resistance in HCC cell lines. Subsequently, we used constitutive form of MKK3/6 (MKK3/6E) to confirm that massive cell death was induced by the activation of p38, and demonstrated the ability to activate p38 without any stimulation. In addition, sorafenib resistance was reduced by the activation of p38. Subsequently, we confirmed that TGF-ß shRNA effectively recovered the phosphorylation of p38 inhibited by sorafenib, and increased the sensitivity of HCC cells to sorafenib, thereby inducing cell death and overcoming the resistance of HCC cells to sorafenib. CONCLUSION: Our study provides a new therapeutic strategy for HCC that overcomes the resistance of HCC to sorafenib by down-regulation of TGF-ß.

Prime-boost immunization by both DNA vaccine and oncolytic adenovirus expressing GM-CSF and shRNA of TGF-ß2 induces anti-tumor immune activation.

A successful DNA vaccine for the treatment of tumors should break established immune tolerance to tumor antigen. However, due to the relatively low immunogenicity of DNA vaccines, compared to other kinds of vaccines using live virus or protein, a recombinant viral vector was used to enhance humoral and cellular immunity. In the current study, we sought to develop a novel anti-cancer agent as a complex of DNA and oncolytic adenovirus for the treatment of malignant melanoma in the C57BL/6 mouse model. MART1, a human melanoma-specific tumor antigen, was used to induce an increased immune reaction, since a MART1-protective response is required to overcome immune tolerance to the melanoma antigen MelanA. Because GM-CSF is a potent inducer of anti-tumor immunity and TGF-ß2 is involved in tumor survival and host immune suppression, mouse GM-CSF (mGM-CSF) and shRNA of mouse TGF-ß2 (shmTGF-ß2) genes were delivered together with MART1 via oncolytic adenovirus. MART1 plasmid was also used for antigen-priming. To compare the anti-tumor effect of oncolytic adenovirus expressing both mGM-CSF and shmTGF-ß2 (AdGshT) with that of oncolytic adenovirus expressing mGM-CSF only (AdG), each virus was intratumorally injected into melanoma-bearing C57BL/6 mice. As a result, mice that received AdGshT showed delayed tumor growth than those that received AdG. Heterologous prime-boost immunization was combined with oncolytic AdGshT and MART1 expression to result in further delayed tumor growth. This regression is likely due to the following 4 combinations: MART1-derived mouse melanoma antigen-specific immune reaction, immune stimulation by mGM-CSF/shmTGF-ß2, tumor growth inhibition by shmTGF-ß2, and tumor cell-specific lysis via an oncolytic adenovirus. Immune activation was mainly induced by mature tumor-infiltrating dendritic cell (TIDC) and lowered regulatory T cells in tumor-infiltrating lymphocytes (TIL). Taken together, these findings demonstrate that human MART1 induces a mouse melanoma antigen-specific immune reaction. In addition, the results also indicate that combination therapy of MART1 plasmid, together with an oncolytic adenovirus expressing MART1, mGM-CSF, and shmTGF-ß2, is a promising candidate for the treatment of malignant melanoma.

Survivin silencing and TRAIL expression using oncolytic adenovirus increase anti-tumorigenic activity in gemcitabine-resistant pancreatic cancer cells.

In this study, we demonstrated that survivin downregulation with TRAIL expression greatly enhanced the cytotoxic death of pancreatic cancer cells after gemcitabine treatment. Using real-time RT-PCR, we analyzed five survivin shRNAs to identify the best target sequence for suppression of human survivin, with the goal of treating gemcitabine-resistant pancreatic cancer cells. Survivin shRNA 5, corresponding to target 5, showed the greatest reduction in survivin mRNA levels. Furthermore, combined treatment with survivin shRNA-expressing adenovirus with gemcitabine plus TRAIL decreased uncleaved PARP and increased consequent PARP cleavage, which was correlated with the greatest levels of survivin downregulation and cell death. These results indicate that survivin functions as a common mediator of gemcitabine- and TRAIL-induced cell death. Using a nude mouse model implanted with MiaPaCa-2 pancreatic cancer cells, we observed tumor regression induced by an oncolytic adenovirus expressing survivin shRNA and TRAIL plus gemcitabine. Together, our findings provide a strong rationale for treating pancreatic cancer patients with both gemcitabine and oncolytic adenovirus armed with survivin shRNA and TRAIL.

Silencing Daxx increases the anti-tumor activity of a TRAIL/shRNA Bcl-xL-expressing oncolytic adenovirus through enhanced viral replication and cellular arrest.

We previously showed that an increase of cellular Bcl-xL mediates acquired resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and knockdown of Bcl-xL expression greatly sensitized TRAIL-induced cytotoxicity. Here, we show that Daxx downregulation increases the anti-tumorigenic activity through enhancement of viral replication and cellular arrest with combination of TRAIL/shBcl-xL-induced apoptosis. This study was conducted to determine the effect of Daxx downregulation on the anti-tumorigenesis induced by oncolytic adenovirus arming TRAIL or TRAIL/shRNA of Bcl-xL genes. Unlike the enhanced cancer cell death induced by exogenous TRAIL or TRAIL plus shRNA of Bcl-xL, oncolytic adenovirus expressing TRAIL or TRAIL plus shRNA of Bcl-xL did not show much enhanced cancer cell death compared to oncolytic adenovirus itself. On the other hand, enhanced cytotoxic cell death and viral replication was observed after infection with oncolytic adenovirus expressing TRAIL plus shRNA of Bcl-xL and shRNA of Daxx at the same construct. Then we realized that enhanced adenoviral replication through Daxx downregulation was caused by increased adenoviral E1A protein expression and Daxx downregulation also stimulated cellular arrest through p21/p53 accumulation. Taken all together, we have shown here that Daxx downregulation should be essentially needed for the increase of anti-tumor activity through enhancement of viral replication and cellular arrest with the combination of TRAIL/shBcl-xL-induced apoptosis and oncolytic adenovirus.

Ratio of phosphorylated HSP27 to nonphosphorylated HSP27 biphasically acts as a determinant of cellular fate in gemcitabine-resistant pancreatic cancer cells.

Gemcitabine has been used most commonly as an anticancer drug to treat advanced pancreatic cancer patients. However, intrinsic or acquired resistance of pancreatic cancer to gemcitabine was also developed, which leads to very low five-year survival rates. Here, we investigated whether cellular levels of HSP27 phosphorylation act as a determinant of cellular fate with gemcitabine. In addition we have demonstrated whether HSP27 downregulation effectively could overcome the acquisition of gemcitabine resistance by using transcriptomic analysis. We observed that gemcitabine induced p38/HSP27 phosphorylation and caused acquired resistance. After acquisition of gemcitabine resistance, cancer cells showed higher activity of NF-κB. NF-κB activity, as well as colony formation in gemcitabine-resistant pancreatic cancer cells, was significantly decreased by HSP27 downregulation and subsequent TRAIL treatment, showing that HSP27 was a common network mediator of gemcitabine/TRAIL-induced cell death. After transcriptomic analysis, gene fluctuation after HSP27 downregulation was very similar to that of pancreatic cancer cells susceptible to gemcitabine, and then in opposite position to that of acquired gemcitabine resistance, which makes it possible to downregulate HSP27 to overcome the acquired gemcitabine resistance to function as an overall survival network inhibitor. Most importantly, we demonstrated that the ratio of phosphorylated HSP27 to nonphosphorylated HSP27 rather than the cellular level of HSP27 itself acts biphasically as a determinant of cellular fate in gemcitabine-resistant pancreatic cancer cells.

Establishment of a mouse melanoma model system for the efficient infection and replication of human adenovirus type 5-based oncolytic virus.

Due to poor adenoviral infectivity and replication in mouse tumor cell types compared with human tumor cell types, use of human-type adenoviral vectors in mouse animal model systems was limited. Here, we demonstrate enhanced infectivity and productive replication of adenovirus in mouse melanoma cells following introduction of both the Coxsackievirus and adenovirus receptor (CAR) and E1B-55K genes. Introduction of CAR into B16BL6 or B16F10 cells increased the infectivity of GFP-expressing adenovirus; however, viral replication was unaffected. We demonstrated a dramatic increase of adenoviral replication (up to 100-fold) in mouse cells via E1B-55K expression and subsequent viral spreading in mouse tissue. These results reveal for the first time that human adenovirus type 5 (Ad5)-based oncolytic virus can be applied to immunocompetent mouse with the introduction of CAR and E1B-55K to syngenic mouse cell line.

The effectiveness of the oncolytic activity induced by Ad5/F35 adenoviral vector is dependent on the cumulative cellular conditions of survival and autophagy.

To overcome the poor tumor transduction efficiency of adenovirus serotype 5 (Ad5) observed in several types of cancer, the fiber region of Ad5, apart from its tail, was replaced by adenovirus serotype 35 (Ad35). The chimeric Ad5/F35 adenoviral vector did not exhibit any significant enhancement of transduction efficiency. CD46, a receptor for Ad35, was expressed in relatively small amounts in most of the cancer cells examined. Therefore, we investigated the pivotal factor(s) that render cancer cells susceptible to transduction. We discovered that the tumor transduction efficiency of Ad5/F35 was enhanced in the presence of rapamycin, an autophagy inducer, in some cancer cells. Analysis of survival potential and cell proliferation rates revealed that Ad5/F35 exerted a more pronounced oncolytic effect in cancer cells with higher survival potential in the presence of rapamycin.

Crosstalk from survival to necrotic death coexists in DU-145 cells by curcumin treatment.

Curcumin as an anticancer agent was investigated in regards to its ability to regulate the switching of cancer cells from survival to necrotic cell death. At higher concentrations, curcumin induced ROS production leading to JNK and p38 phosphorylation in DU-145 prostate cancer cells. Of the MAP kinases, ERK or p38/JNK were phosphorylated earlier during curcumin treatment, and were responsible for curcumin-induced cell survival at early time of treatment with the help of phosphorylated Akt, while significant amounts of ROS production in later periods stimulated cell death with caspase degradation. In addition to autophagic signaling, necrosis was dominant with little apoptotic cell death. Caspase activation was completely prohibited by procaspase degradation, which contributed to curcumin-induced early necrosis. At the later incubation period (24h), cytotoxicity caused by curcumin peaked, at which time survival or proliferation signals, such as phosphorylated Akt and phosphorylated ERK, was almost completely diminished. Curcumin-induced ROS were shown to function, biphasically depending on the incubation period; facilitating survival, in the earlier incubation period, and necrotic death in the later. Based on all of these results, we concluded that curcumin contributes to a complex signaling network, affecting cell survival and necrotic cell death, which in turn could inhibit apoptotic cell death.

TRAIL/MEKK4/p38/HSP27/Akt survival network is biphasically modulated by the Src/CIN85/c-Cbl complex.

Previously, we showed that mitogen-activated protein kinase/extracellular signal-related kinase 4 (MEKK4) is responsible for p38 activation and that its activation during tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment also increases the catalytic activity of Akt. Here, we further investigated how the TRAIL-induced MEKK4/p38/heat shock protein (HSP27)/Akt survival network is modulated by the Src/c-Cbl interacting protein of 85kDa (CIN85)/c-Cbl complex. TRAIL-induced activation of Akt catalytic activity and phosphorylation were highly correlated with p38/HSP27 phosphorylation, whereas the phosphorylation of p38/HSP27 increased further during incubation with curcumin and TRAIL, which caused significant apoptotic cell death. CIN85, a c-Cbl-binding protein, plays an essential role in connecting cell survival to cell death. The interaction of CIN85 with MEKK4 was increased during the late phase of TRAIL incubation, suggesting that sustained p38 and HSP27 phosphorylation protects cells by preventing further cell death. However, further increases in p38/HSP27 phosphorylation induced by cotreatment with curcumin and TRAIL converted cell fate to death. Taken together, these data demonstrate that phosphorylated p38/HSP27 as biphasic modulators act in conjunction with CIN85 to determine whether cells survive or die in response to apoptotic stress.

HSP27 modulates survival signaling networks in cells treated with curcumin and TRAIL.

The combination of curcumin and TRAIL and their role in enhancing apoptotic cell death has been reported by many studies. However, the exact molecular mechanism of apoptosis mediated by curcumin and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is not yet completely understood. In this study, we observed a close connection between dephosphorylated Akt and an increase in phosphorylated heat shock protein 27 (HSP27) during combined treatment with curcumin and TRAIL. Akt dephosphorylation was cumulatively regulated by protein phosphatase 1 (PP1), phosphoinositide-dependent kinase-1 (PDK1), and src. PP1 and PDK1 directly interacted with HSP27, whereas src indirectly interacted with HSP27 via the tumor necrosis factor receptor-associated factor 6 complex. In conclusion, HSP27 modulated cell survival by its interactions with various binding partners, depending on the level of phosphorylated HSP27.

MEKK1/MEKK4 are responsible for TRAIL-induced JNK/p38 phosphorylation.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to activate mitogen-activated protein kinases (MAPKs) depending on caspase and mammalian sterile 20-like kinase 1 activations. However, the upstream molecule of MAPKs has not yet been identified. The mitogen-activated protein kinase kinase 1 (MEKK1) and the apoptosis signal-regulating kinase 1 (ASK1) are considered to be possible candidates for the action of MAPKKKs induced by TRAIL and the possibility of reactive oxygen species involvement has also been investigated. We found that MEKK1/MEKK4 as opposed to ASK1, are responsible for TRAIL-induced c-Jun NH2-terminal kinase (JNK) or p38 activation, and that their catalytic activity is repressed by the caspase-8 inhibitor, suggesting that the caspase-8 activation induced by TRAIL is indispensible for MEKK activation. The 14-3-3 θ was also shown to interact with and to dissociate from MEKK1 by TRAIL treatment, thus implicating the 14-3-3 protein as a negative regulator of MEKK1 activation. Taken together, we show herein that the upstream molecule of the TRAIL-induced MAPK activation is MEKK, as opposed to ASK1, via the mediation of its signal through JNK/p38 in a caspase-8-dependent manner.

TRAIL-induced caspase/p38 activation is responsible for the increased catalytic and invasive activities of Akt.

We previously observed that TRAIL induces acquired TRAIL resistance coinciding with increased Akt phosphorylation brought about by the Src-PI3K-Akt signaling pathways and mediated by c-Cbl. c-Cbl, a ubiquitously expressed cytoplasmic adaptor protein, is simultaneously involved in the rapid degradation of TRAIL receptors and Akt phosphorylation during TRAIL treatment. Here, we show that Akt phosphorylation is not exclusively responsible for acquired TRAIL resistance. Akt catalytic activation is known to increase during metabolic oxidative stress, but we show that TRAIL also dramatically induces the catalytic activation of Akt in TRAIL-sensitive cells, but not in TRAIL-resistant cells. This suggests that Akt catalytic activation during TRAIL-induced apoptosis is likely to play a compensatory role in the maintenance of cell homeostasis. In addition, activated p38 and phosphorylated HSP27 were found to act as downstream effector molecules of p38 during TRAIL treatment and were shown to be responsible for increased Akt catalytic and invasive activities.

c-Cbl-mediated degradation of TRAIL receptors is responsible for the development of the early phase of TRAIL resistance.

We previously reported two modes of development of acquired TRAIL resistance: early phase and late phase [1]. In these studies, we observed that greater Akt activity and the expression of Bcl-xL were related mainly to the late phase of acquired TRAIL resistance. Recently we became aware of a possible mechanism of early phase TRAIL resistance development through internalization and degradation of TRAIL receptors (DR4 and DR5). Our current studies demonstrate that TRAIL receptors rapidly diminish at the membrane as well as the cytoplasm within 4h after TRAIL exposure, but recover completely after one or two days. Our studies also reveal that Cbl, a ubiquitously expressed cytoplasmic adaptor protein, is responsible for the rapid degradation of TRAIL receptors; Cbl binds to them and induces monoubiquitination of these receptors concurrent with their degeneration soon after TRAIL exposure, creating the early phase of acquired TRAIL resistance.

c-Cbl acts as a mediator of Src-induced activation of the PI3K-Akt signal transduction pathway during TRAIL treatment.

We have previously observed that TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) induces acquired TRAIL resistance by increasing Akt phosphorylation and Bcl-xL expression. In this study, we report that Src, c-Cbl, and PI3K are involved in the phosphorylation of Akt during TRAIL treatment. Data from immunoprecipitation and immunoblotting assay reveal that Src interacts with c-Cbl and PI3K. Data from immune complex kinase assay demonstrate that Src can directly phosphorylate c-Cbl and PI3K p85 subunit protein. Data from gene knockdown experiments with an RNA interference (RNAi) technique show that c-Cbl is involved in the interaction between Src and PI3K p85 during TRAIL treatment, playing an important role in TRAIL-induced Akt phosphorylation. Taken together, c-Cbl may act as a mediator to regulate the Src-PI3K-Akt signal transduction pathway during TRAIL treatment.

Simultaneous improvement of catalytic activity and thermal stability of tyrosine phenol-lyase by directed evolution.

The tyrosine phenol-lyase from Symbiobacterium toebii was engineered to improve both its stability and catalytic activity by the application of random mutagenesis and subsequent reassembly of the acquired mutations. Activity screening of the random library produced four mutants with a two-fold improved activity, whereas parallel screening after heat treatment at 65 degrees C identified three mutants with half-inactivation temperatures improved by up to 5.6 degrees C. The selected mutants were then reassembled using the staggered extension PCR method, and subsequent screening of the library produced seven mutants with up to three-fold improved activity and half-inactivation temperatures improved by up to 11.2 degrees C. Sequence analyses revealed that the stability-improved hits included A13V, E83K and T407A mutations, whereas the activity-improved hits included the additional T129I or T451A mutation. In particular, the A13V mutation was propagated in the hits with improved stability during the reassembly-screening process, indicating the critical nature of the N-terminal moiety for enzyme stability. Furthermore, homology modeling of the enzyme structure revealed that most of the stability mutations were located around the dimer-dimer interface, including the N-terminus, whereas the activity-improving mutations were located further away, thereby minimizing any interference that would be detrimental to the co-improvement of the stability and catalytic activity of the enzyme.