Shibin Zhou - Academia.edu (original) (raw)

Papers by Shibin Zhou

The Journal of Neuroscience, 1992

The Fi-20 protein is a novel neuronal-specific, synapseassociated protein that is expressed nonun... more The Fi-20 protein is a novel neuronal-specific, synapseassociated protein that is expressed nonuniformly in mouse brain. Expression of the Fl-20 protein is developmentally regulated in a pattern coincident with active synaptogenesis and synaptic maturation. Here we report the cloning of the cDNA sequence for the Fl-20 protein. We found two distinct isoforms of Fl-20 cDNA that differed by the presence of 15 additional nucleotides, which does not interrupt the open reading frame. RNase protection analysis and PCR amplification of mouse brain RNA revealed that both isoforms are present in cellular RNA. It is likely that the two Fl-20 mRNA isoforms are derived from RNA splicing events utilizing alternative 3' acceptor sites. Analysis of the deduced amino acid sequence for the complete open reading frame revealed that the predominant Fl-20 mRNA encodes an 898 amino acid polypeptide with a molecular weight of 91,319 Da. The deduced amino acid sequence does not contain a signal sequence, or any extensive hydrophobic regions. The deduced amino acid sequence does contain a number of consensus sequences for protein kinases. Searches of the protein and nucleic acid sequence data bases revealed that the Fl-20 protein has not been previously characterized at the primary structure level, although a weak similarity was found between rabbit calpastatin and the C-terminal portion of the Fl-20 protein. We then determined biochemically that the Fl-20 protein is a substrate for Ca2+-dependent proteolysis, which is specifically inhibited by calpain inhibitors in vitro. This indicates that the Fl-20 protein is a substrate for neuronal calpain. We observed that treatment of a synaptosomal lysate with alkaline phosphatase led to an increase in the electrophoretic mobility of the Fl-20 protein, as well as to

Cancer Research, 2015

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Most cancer drug... more Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Most cancer drugs have a narrow therapeutic window where dose is dictated by toxicity and, in turn, causes decreased efficacy. Adverse side effects are the primary cause of early cessation of chemotherapy as well as high failure rates of new entities during clinical trials. This has led to intense research into various tumor-selective delivery modalities. We present here two novel strategies by which we reformulate promising cancer drug candidates and dramatically lower their toxic side effects. The first strategy involved development of a generalizable method for efficiently loading poorly soluble, non-ionizible organic molecules into stealth liposomes using a pH gradient. Using this new methodology we successfully encapsulated the PLK-1 inhibitor, BI-2536 and the MEK-1 inhibitor, PD-0325901, both of which had failed Phase II despite exceptional promise in preclinical and Phase I stages. In the second case we successfully packaged the potent anti-glycolytic alkylating agent, 3-bromopyruvate, previously used only under loco-regional therapy settings, for systemic therapy and demonstrated its exceptional promise in an orthotopic xenograft model of pancreatic cancer. Together, our data presented below demonstrate innovative strategies for opening up the therapeutic window of toxic cancer drugs. Our first approach uses drug-packaged stealth However, liposomal formulations have not gained widespread use because many drugs are highly hydrophobic and non-ionizable, and as such, cannot be efficiently loaded into liposomes through established passive or active techniques. To overcome these issues we designed a generalized strategy that have three important features: 1) pH gradient across liposome bilayer for directional loading of drugs, 2) use of β-cyclodextrins to create solubilize hydrophobic drugs regardless of the physico-chemical properties of the agents themselves, 3) synthetic analogs of β-cyclodextrins containing multiple weakly basic or weakly acidic functional groups on their solvent exposed surfaces to trap the cyclodextrin-drug complexes in liposomes, exploiting the ionizable groups on the cyclodextrins, rather than on the drugs themselves. First, we established that cyclodextrins labelled with a fluorescent moiety, but without a drug payload, accumulated in liposomes containing acidic citrate buffer with >90% loading efficiency compared to minimal accumulation in neutral liposomes at pH 7.4 without the pH gradient. These data suggests that the cyclodextrins can be transported across the lipid membrane and are trapped within the aqueous milieu. We next tested if the modified cyclodextrins could ferry and trap hydrophobic compounds within the liposome using common hydrophobic organic dyes (coumarins) to determine whether the modified cyclodextrins could ferry hydrophobic compounds across the liposome lipid bilayer. All cyclodextrin-coumarin complexes were incorporated into liposomes with high efficiency (>95%). In contrast, coumarins in the absence of cyclodextrins and unmodified cyclodextrin-coumarins complexes were poorly incorporated into liposomes under identical conditions. These results establish that ionizable cyclodextrins not only cross the lipid bilayer, they are able to carry a non-ionizable payload with them. We next investigated the ability of functionalized cyclodextrins to load into liposomes anti neoplastic drug candidates that had failed Phase II human trials. For this purpose we chose 1) BI-2536 (Boehringer Ingelheim), a highly selective inhibitor of PLK1, and 2) PD-0325901 (Pfizer), an allosteric MEK-1 inhibitor as our “rescue” candidates. BI-2536 was the subject of several clinical trials in patients with cancers of the lung, breast, ovaries, and uterus. Although BI-2536 showed evidence of efficacy in cancer patients, development was abandoned after Phase II trials revealed unacceptable toxicity (grade 4 neutropenia) at sub-therapeutic doses. Similarly, PD-0325901 was the subject of multiple clinical trials before being abandoned after Phase II trials due to retinal vein occlusion. We hypothesized that packing of these drugs in liposomes would reduce normal tissue exposure and mitigate the clinical toxicities observed. Using our new methodology we were able to reproducibly load both BI-2536-cyclodextrin and PD-0325901-cyclodextrin complexes into liposomes, achieving stable aqueous solutions containing 10 mg/ml and 5mg/ml of the drugs respectively. We, then, assessed their effects in nude mice bearing subcutaneous xenografts of colon cancer cells. The CYCL version of both drugs proved far superior to the corresponding free forms with respect to both toxicity and efficacy. While the free drugs exhibited both acute and delayed toxicity in our murine models, the CYCL-drugs did not ellicit any noticeable adverse reactions even at doses far greater than the MTD. In three different xenograft models, not only…

Proceedings of the National Academy of Sciences, 2015

Significance Mutations in oncogenes and tumor suppressor genes drive tumorigenesis and their prot... more Significance Mutations in oncogenes and tumor suppressor genes drive tumorigenesis and their protein products form therapeutic targets that are absent from normal cells. However, nearly all such mutant epitopes lie in the interior of the cells, either in the cytoplasm or nucleus, complicating immunotherapies directed against the mutants. We have developed an approach to identify antibodies selectively targeting complexes containing common HLA types bound to peptide products of commonly mutated oncogenes. Because these peptide-HLA complexes are expected to be exclusively present on the surface of cancer cells, antibodies targeting them could in principle be used for therapeutic purposes.

Oncotarget, Jan 20, 2015

Glioblastoma (GBM) is a highly aggressive primary brain tumor that is especially difficult to tre... more Glioblastoma (GBM) is a highly aggressive primary brain tumor that is especially difficult to treat. The tumor's ability to withstand hypoxia leads to enhanced cancer cell survival and therapy resistance, but also yields a microenvironment that is in many aspects unique within the human body, thus offering potential therapeutic opportunities. The spore-forming anaerobic bacterium Clostridium novyi-NT(C. novyi-NT) has the ability to propagate in tumor-generated hypoxia, leading to oncolysis. Here, we show that intravenously injected spores of C. novyi-NT led to dramatic tumor destructions and significant survival increases in implanted, intracranial syngeneic F98 and human xenograft 060919 rat GBM models. C. novyi-NT germination was specific and confined to the neoplasm, with sparing of the normal brain parenchyma. All animals tolerated the bacteriolytic treatment, but edema and increased intracranial pressure could quickly be lethal if not monitored and medically managed with hy...

Oncotarget

While nanoparticles have shown great promise as drug carriers in cancer therapy, their effectiven... more While nanoparticles have shown great promise as drug carriers in cancer therapy, their effectiveness is critically dependent on the structural characteristics of the tumor vasculature. Here we demonstrate that several agents capable of inducing vascular responses akin to those observed in inflammatory processes enhance the accumulation of nanoparticles in tumors. The vascular-active agents tested in this study included a bacterium, a pro-inflammatory cytokine, and microtubule-destabilizing drugs. Using radiolabeled nanoparticles, we show that such agents can increase the tumor to blood ratio of radioactivity by more than 20-fold compared to nanoparticles alone. Moreover, vascular-active agents dramatically improved the therapeutic effect of nanoparticles containing radioactive isotopes or chemotherapeutic agents. This resulted in cures of animals with subcutaneous tumors and significantly prolonged the survival of animals with orthotopic brain tumors. In principle, a variety of vasc...

Oncotarget, 2011

Tumor necrosis factor-α (TNF-α) has been discussed as a potential anticancer agent for many years... more Tumor necrosis factor-α (TNF-α) has been discussed as a potential anticancer agent for many years, however initial enthusiasm about its clinical use as a systemic agent was curbed due to significant toxicities and lack of efficacy. Combination of TNF-α with chemotherapy in the setting of hyperthermic isolated limb perfusion (ILP), has provided new insights into a potential therapeutic role of this agent. The therapeutic benefit from TNF-α in ILP is thought to be not only due to its direct anti-proliferative effect, but also due to its ability to increase penetration of the chemotherapeutic agents into the tumor tissue. New concepts for the use of TNF-α as a facilitator rather than as a direct actor are currently being explored with the goal to exploit the ability of this agent to increase drug delivery and to simultaneously reduce systemic toxicity. This review article provides a comprehensive overview on the published previous experience with systemic TNF-α. Data from 18 phase I an...

Oncotarget, 2010

Previous genetic analyses have suggested that mutations of the genes encoding PI3Kα facilitate in... more Previous genetic analyses have suggested that mutations of the genes encoding PI3Kα facilitate invasion and metastasis but have less effect on primary tumor growth. These findings have major implications for therapeutics but have not been factored into pre-clinical drug development designs. Here we show that the inhibition of PI3Kα by newly designed small molecule inhibitors prevented metastasis formation in mice but had much less effect on the growth of subcutaneous xenografts or primary intra-abdominal tumors. These data support the idea that PI3Kα plays an important role in the metastatic process and suggest a more informed strategy for selecting drugs worthy of further development for clinical application.

Science translational medicine, Jan 13, 2014

Species of Clostridium bacteria are notable for their ability to lyse tumor cells growing in hypo... more Species of Clostridium bacteria are notable for their ability to lyse tumor cells growing in hypoxic environments. We show that an attenuated strain of Clostridium novyi (C. novyi-NT) induces a microscopically precise, tumor-localized response in a rat orthotopic brain tumor model after intratumoral injection. It is well known, however, that experimental models often do not reliably predict the responses of human patients to therapeutic agents. We therefore used naturally occurring canine tumors as a translational bridge to human trials. Canine tumors are more like those of humans because they occur in animals with heterogeneous genetic backgrounds, are of host origin, and are due to spontaneous rather than engineered mutations. We found that intratumoral injection of C. novyi-NT spores was well tolerated in companion dogs bearing spontaneous solid tumors, with the most common toxicities being the expected symptoms associated with bacterial infections. Objective responses were obser...

Science, 2013

Over the past decade, comprehensive sequencing efforts have revealed the genomic landscapes of co... more Over the past decade, comprehensive sequencing efforts have revealed the genomic landscapes of common forms of human cancer. For most cancer types, this landscape consists of a small number of "mountains" (genes altered in a high percentage of tumors) and a much larger number of "hills" (genes altered infrequently). To date, these studies have revealed ~140 genes that, when altered by intragenic mutations, can promote or "drive" tumorigenesis. A typical tumor contains two to eight of these "driver gene" mutations; the remaining mutations are passengers that confer no selective growth advantage. Driver genes can be classified into 12 signaling pathways that regulate three core cellular processes: cell fate, cell survival, and genome maintenance. A better understanding of these pathways is one of the most pressing needs in basic cancer research. Even now, however, our knowledge of cancer genomes is sufficient to guide the development of more effective approaches for reducing cancer morbidity and mortality. Ten years ago, the idea that all of the genes altered in cancer could be identified at base-pair resolution would have seemed like science fiction. Today, such genome-wide analysis, through sequencing of the exome (see Box 1, Glossary, for definitions of terms used in this Review) or of the whole genome, is routine. Box 1 Glossary Adenoma A benign tumor composed of epithelial cells. Alternative lengthening of telomeres (ALT) A process of maintaining telomeres independent of telomerase, the enzyme normally responsible for telomere replication. Amplification A genetic alteration producing a large number of copies of a small segment (less than a few megabases) of the genome. Angiogenesis the process of forming vascular conduits, including veins, arteries, and lymphatics.

Science, 2006

Clostridium novyi-NT is an anaerobic bacterium that can infect hypoxic regions within experimenta... more Clostridium novyi-NT is an anaerobic bacterium that can infect hypoxic regions within experimental tumors. Because C. novyi-NT lyses red blood cells, we hypothesized that its membrane-disrupting properties could be exploited to enhance the release of liposome-encapsulated drugs within tumors. Here, we show that treatment of mice bearing large, established tumors with C. novyi-NT plus a single dose of liposomal doxorubicin often led to eradication of the tumors. The bacterial factor responsible for the enhanced drug release was identified as a previously unrecognized protein termed liposomase. This protein could potentially be incorporated into diverse experimental approaches for the specific delivery of chemotherapeutic agents to tumors.

Proceedings of the National Academy of Sciences, 2005

Bacterial infections provide diagnostic dilemmas that could be enlightened by modern imaging tech... more Bacterial infections provide diagnostic dilemmas that could be enlightened by modern imaging technologies. We have developed a simple method for imaging bacterial infections in mice that relies on the phosphorylation and trapping of the thymidine kinase (TK) substrate 1-(2′-deoxy-2′-fluoro-β- d -arabinofuranosyl)-5-[ 125 I] iodouracil ([ 125 I]FIAU) within bacteria. FIAU was found to inhibit the growth of WT Escherichia coli but not a TK – strain, indicating that WT E. coli could metabolize this compound. In silico analyses demonstrated that all pathogenic strains of bacteria whose genomes have been sequenced contain a TK gene highly homologous to the E. coli TK. Accordingly, we demonstrated that localized infections caused by representatives of five genera of bacteria could be readily imaged with [ 125 I]FIAU. Such imaging provides a general method for the diagnosis of localized bacterial infections that could be translatable to the clinic.

Proceedings of the National Academy of Sciences, 1998

Smad4 ( DPC4 ) is a candidate tumor suppressor gene that has been hypothesized to be critical for... more Smad4 ( DPC4 ) is a candidate tumor suppressor gene that has been hypothesized to be critical for transmitting signals from transforming growth factor (TGF) β and related ligands. To directly test this hypothesis, the Smad4 gene was deleted through homologous recombination in human colorectal cancer cells. This deletion abrogated signaling from TGF-β, as well as from the TGF-β family member activin. These results provide unequivocal evidence that mutational inactivation of Smad4 causes TGF-β unresponsiveness and provide a basis for understanding the physiologic role of this gene in tumorigenesis.

Nature Genetics, 2011

Through exomic sequencing of ten hepatitis C virus (HCV)-associated hepatocellular carcinomas (HC... more Through exomic sequencing of ten hepatitis C virus (HCV)-associated hepatocellular carcinomas (HCC) and subsequent evaluation of additional affected individuals, we discovered novel inactivating mutations of ARID2 in four major subtypes of HCC (HCV-associated HCC, hepatitis B virus (HBV)-associated HCC, alcohol-associated HCC and HCC with no known etiology). Notably, 1 8.2% of individuals with HCV-associated HCC in the United States and Europe harbored ARID2 inactivation mutations, suggesting that ARID2 is a tumor suppressor gene that is relatively commonly mutated in this tumor subtype. With an estimated 748,000 newly diagnosed cases per year, HCC is the third leading cause of cancer-related deaths worldwide 1. In the United States, the five-year survival rate of individuals with liver cancer is 11.7%, making it one of the most lethal forms of neoplasia 2. Epidemiologic studies have conclusively linked HBV and HCV infections as well as alcohol consumption and aflatoxin B exposure to the development of HCC 3. However, whether

Nature Biotechnology, 2006

Bacteriolytic anti-cancer therapies employ attenuated bacterial strains that selectively prolifer... more Bacteriolytic anti-cancer therapies employ attenuated bacterial strains that selectively proliferate within tumors. Clostridium novyi-NT spores represent one of the most promising of these agents, as they generate potent anti-tumor effects in experimental animals. We have determined the 2.55-Mb genomic sequence of C. novyi-NT, identifying a new type of transposition and 139 genes that do not have homologs in other bacteria. The genomic sequence was used to facilitate the detection of transcripts expressed at various stages of the life cycle of this bacterium in vitro as well as in infections of tumors in vivo. Through this analysis, we found that C. novyi-NT spores contained mRNA and that the spore transcripts were distinct from those in vegetative forms of the bacterium.

Molecular Cell, 1998

genes identified as critical mediators of TGF␤-like sig-and Scott E. Kern † naling (Sekelsky et a... more genes identified as critical mediators of TGF␤-like sig-and Scott E. Kern † naling (Sekelsky et al., 1995; Savage et al., 1996). In * Molecular Genetics Laboratory vertebrate cells, Smads 1, 2, 3, and 5 are phosphorylated The Johns Hopkins Oncology Center by activated RI receptors at a conserved SSXS motif Baltimore, Maryland 21231 located at their carboxyl termini, whereupon they trans-† Department of Pathology and locate from the cell membrane or cytoplasm to the nu-The Oncology Center cleus (

Molecular Cell, 1998

teins (Liu et al., 1996). Two ways in which Smad activation could lead to transcriptional activat... more teins (Liu et al., 1996). Two ways in which Smad activation could lead to transcriptional activation have been Johns Hopkins Oncology Center Baltimore, Maryland 21231 identified. First, it has been shown that human Smad3 and Smad4, but not Smad2, can bind to specific DNA sequences and activate transcription of adjacent reporters (Zawel et al., 1998). A similar sequence-specific ac-Summary tivity is present in Drosophila Mad (Kim et al., 1997). Second, Smad2 has been shown to bind to the Xenopus We have identified a human homolog of the Xenopus laevis forkhead protein FAST-1 (xFAST-1) and particiforkhead activin signal transducer-1 (xFAST-1). Alpate in a complex exhibiting sequence-specific binding though significantly different in sequence from its Xenactivity attributable to the xFAST-1 component (Chen opus counterpart, hFAST-1 shared with xFAST-1 the et al., 1996, 1997; Liu et al., 1997). Although Smad4 does ability to bind to human Smad2 and activate an activin not directly bind to xFAST-1, Smad4 is recruited to the response element (ARE). The hFAST-1-dependent ac-xFAST-1/Smad2 complex by Smad2 (Chen et al., 1997; tivation of ARE was completely dependent on endoge-Liu et al., 1997). nous Smad4 and stimulation by a TGF␤-like ligand. TGF␤-like responses are remarkably widespread in The hFAST-1 protein was shown to bind to a novel eukaryotes and are important not only in development DNA motif, TGT (G/T) (T/G)ATT, an exact copy of which but also in cancer (Fynan and Reiss, 1993; Hartsough was present within the ARE. A single copy of this motif and Mulder, 1997). Further progress in understanding could activate a reporter in a TGF␤-dependent fashion the varied developmental and oncogenic ramifications but only when an adjacent Smad-binding element was of these pathways in mammalian cells will depend on the present in the construct. These data suggest that reidentification and analysis of the relevant mammalian sponses to TGF␤ family members may be mediated by genes. We here report the identification and characa DNA-binding complex formed by hFAST-1, hSmad2, terization of a human FAST-1 homolog. Though signifiand hSmad4. cantly different in sequence from the Xenopus prototype, hFAST-1 retains the ability to mediate transcriptional

Magnetic Resonance in Medicine, 2013

Purpose-To develop a non-invasive MRI method for determining the germination and infection of tum... more Purpose-To develop a non-invasive MRI method for determining the germination and infection of tumor-homing bacteria in bacteriolytic cancer therapy using endogenous CEST contrast. Methods-The CEST parameters of the anaerobic gram-positive bacterium Clostridium novyi-NT (C. novyi-NT) were first characterized in vitro, then used to detect C. novyi-NT germination and infection in subcutaneous CT26 colorectal tumor-bearing mice (n=6) after injection of 300 million bacterial spores. Lipopolysacharide (LPS) injected mice were used to exclude that the changes of CEST MRI were due to inflammation. Results-CEST contrast was observed over a broad frequency range for bacterial suspensions in vitro, with the maximum contrast around 2.6 ppm from the water resonance. No signal could be detected for bacterial spores, demonstrating the specificity for germination. In vivo, a significant elevation of CEST contrast was identified in C. novyi-NT infected tumors as compared to those before bacterial germination and infection (p<0.05, n=6). No significant change was observed in tumors with LPS-induced sterile inflammation (p> 0.05, n=4). Conclusions-Endogenous bacterial CEST contrast (bacCEST) can be used to monitor the germination and proliferation of the therapeutic bacterium C. novyi-NT without a need for exogenous cell labeling probes.

Journal of Controlled Release, 2014

Nanocarrier-based chemotherapy allows preferential delivery of therapeutics to tumors and has bee... more Nanocarrier-based chemotherapy allows preferential delivery of therapeutics to tumors and has been found to improve the efficacy of cancer treatment. However, difficulties in tracking nanocarriers and evaluating their pharmacological fate in patients have limited judicious selection of patients to those who might most benefit from nanotherapeutics. To enable the monitoring of nanocarriers in vivo, we developed MRI-traceable diamagnetic Chemical Exchange Saturation Transfer (diaCEST) liposomes. The diaCEST liposomes were based on the clinical formulation of liposomal doxorubicin (i.e. DOXIL ®) and were loaded with barbituric acid (BA), a small, organic, biocompatible diaCEST contrast agent. The optimized diaCEST liposomal formulation with a BA

Journal of Bacteriology, 2007

Spores of the anaerobic bacterium Clostridium novyi NT are able to germinate in and destroy hypox... more Spores of the anaerobic bacterium Clostridium novyi NT are able to germinate in and destroy hypoxic regions of tumors in experimental animals. Future progress in this area will benefit from a better understanding of the germination and outgrowth processes that are essential for the tumorilytic properties of these spores. Toward this end, we have used both transmission electron microscopy and atomic force microscopy to determine the structure of both dormant and germinating spores. We found that the spores are surrounded by an amorphous layer intertwined with honeycomb parasporal layers. Moreover, the spore coat layers had apparently self-assembled, and this assembly was likely to be governed by crystal growth principles. During germination and outgrowth, the honeycomb layers, as well as the underlying spore coat and undercoat layers, sequentially dissolved until the vegetative cell was released. In addition to their implications for understanding the biology of C. novyi NT, these st...

The Journal of Neuroscience, 1992

Cancer Research, 2015

Proceedings of the National Academy of Sciences, 2015

Oncotarget, Jan 20, 2015

Oncotarget

Oncotarget, 2011

Oncotarget, 2010

Science translational medicine, Jan 13, 2014

Science, 2013

Science, 2006

Proceedings of the National Academy of Sciences, 2005

Proceedings of the National Academy of Sciences, 1998

Nature Genetics, 2011

Nature Biotechnology, 2006

Molecular Cell, 1998

Magnetic Resonance in Medicine, 2013

Journal of Controlled Release, 2014

Journal of Bacteriology, 2007