Tumour-specific triple-regulated oncolytic herpes virus to target glioma.

Oncolytic herpes simplex virus type 1 (oHSV-1) therapy is an emerging treatment modality that selectively destroys cancer. Here we report use of a glioma specific HSV-1 amplicon virus (SU4-124 HSV-1) to selectively target tumour cells. To achieve transcriptional regulation of the SU4-124 HSV-1 virus, the promoter for the essential HSV-1 gene ICP4 was replaced with a tumour specific survivin promoter. Translational regulation was achieved by incorporating 5 copies of microRNA 124 target sequences into the 3'UTR of the ICP4 gene. Additionally, a 5'UTR of rat fibroblast growth factor -2 was added in front of the viral ICP4 gene open reading frame. Our results confirmed enhanced expression of survivin and eIF4E in different glioma cells and increased micro-RNA124 expression in normal human and mouse brain tissue. SU4-124 HSV-1 had an increased ICP4 expression and virus replication in different glioma cells compared to normal neuronal cells. SU4-124 HSV-1 exerted a strong antitumour effect against a panel of glioma cell lines. Intracranial injection of SU4-124 HSV-1 did not reveal any sign of toxicity on day 15 after the injection. Moreover, a significantly enhanced antitumour effect with the intratumourally injected SU4-124 HSV-1 virus was demonstrated in mice bearing human glioma U87 tumours, whereas viral DNA was almost undetectable in normal organs. Our study indicates that incorporation of multiple cancer-specific regulators in an HSV-1 system significantly enhances both cancer specificity and oncolytic activity.

Targeting unique metabolic properties of breast tumor initiating cells.

Normal stem cells from a variety of tissues display unique metabolic properties compared to their more differentiated progeny. However, relatively little is known about metabolic properties of cancer stem cells, also called tumor initiating cells (TICs). In this study we show that, analogous to some normal stem cells, breast TICs have distinct metabolic properties compared to nontumorigenic cancer cells (NTCs). Transcriptome profiling using RNA-Seq revealed TICs underexpress genes involved in mitochondrial biology and mitochondrial oxidative phosphorylation, and metabolic analyses revealed TICs preferentially perform glycolysis over oxidative phosphorylation compared to NTCs. Mechanistic analyses demonstrated that decreased expression and activity of pyruvate dehydrogenase (Pdh), a key regulator of oxidative phosphorylation, plays a critical role in promoting the proglycolytic phenotype of TICs. Metabolic reprogramming via forced activation of Pdh preferentially eliminated TICs both in vitro and in vivo. Our findings reveal unique metabolic properties of TICs and demonstrate that metabolic reprogramming represents a potential therapeutic strategy for targeting these cells.

Erythropoietin promotes breast tumorigenesis through tumor-initiating cell self-renewal.

Erythropoietin (EPO) is a hormone that induces red blood cell production. In its recombinant form, EPO is the one of most prescribed drugs to treat anemia, including that arising in cancer patients. In randomized trials, EPO administration to cancer patients has been associated with decreased survival. Here, we investigated the impact of EPO modulation on tumorigenesis. Using genetically engineered mouse models of breast cancer, we found that EPO promoted tumorigenesis by activating JAK/STAT signaling in breast tumor-initiating cells (TICs) and promoted TIC self renewal. We determined that EPO was induced by hypoxia in breast cancer cell lines, but not in human mammary epithelial cells. Additionally, we demonstrated that high levels of endogenous EPO gene expression correlated with shortened relapse-free survival and that pharmacologic JAK2 inhibition was synergistic with chemotherapy for tumor growth inhibition in vivo. These data define an active role for endogenous EPO in breast cancer progression and breast TIC self-renewal and reveal a potential application of EPO pathway inhibition in breast cancer therapy.

Neuregulin autocrine signaling promotes self-renewal of breast tumor-initiating cells by triggering HER2/HER3 activation.

Currently, only patients with HER2-positive tumors are candidates for HER2-targeted therapies. However, recent clinical observations suggest that the survival of patients with HER2-low breast cancers, who lack HER2 amplification, may benefit from adjuvant therapy that targets HER2. In this study, we explored a mechanism through which these benefits may be obtained. Prompted by the hypothesis that HER2/HER3 signaling in breast tumor-initiating cells (TIC) promotes self-renewal and survival, we obtained evidence that neuregulin 1 (NRG1) produced by TICs promotes their proliferation and self-renewal in HER2-low tumors, including in triple-negative breast tumors. Pharmacologic inhibition of EGFR, HER2, or both receptors reduced breast TIC survival and self-renewal in vitro and in vivo and increased TIC sensitivity to ionizing radiation. Through a tissue microarray analysis, we found that NRG1 expression and associated HER2 activation occurred in a subset of HER2-low breast cancers. Our results offer an explanation for why HER2 inhibition blocks the growth of HER2-low breast tumors. Moreover, they argue that dual inhibition of EGFR and HER2 may offer a useful therapeutic strategy to target TICs in these tumors. In generating a mechanistic rationale to apply HER2-targeting therapies in patients with HER2-low tumors, this work shows why these therapies could benefit a considerably larger number of patients with breast cancer than they currently reach.

Targeted oncolytic herpes simplex viruses for aggressive cancers.

Herpes simplex virus (HSV) is a well-known vector that is often used for gene therapy to treat cancers. The most attractive feature of HSV is its ability to destroy tumors through a distinctive oncolytic mechanism where the virus can destroy cancer cells via cell lysis, a killing function that no anti-cancer drugs can mimic. Importantly, HSV is a safe and effective virus that can be easily manipulated to preferentially replicate in tumor cells. In the last 20 years of reengineering efforts, a number of HSV designs, including the classical G207, have been focused on deleting viral genes in order to render the virus tumor specific. Although such designs can successfully destroy tumor xenografts in animal models, with minimal impact on normal tissues, a common trade-off is the marked attenuation of the virus. This problem is most profound in many clinical tumors, where virus dissemination is often hindered by the difficult cellular and molecular terrain of the human tumor mass. In order to harness all of HSV's replication potential to destroy tumor cells, efforts in our lab, as well as others, last several years have been focused on engineering an oncolytic HSV to target tumor cells without deleting any viral genes, and have since generated highly tumor specific viruses including our transcriptional translational dually regulated HSV (TTDR-HSV). In this review, we will discuss the improvements associated with the newer TTDR-HSV design compared to the classical defective HSV designs such as G207 and tk- HSV. Lastly, we will review additional cellular features of aggressive tumors, such as their immense cellular heterogeneity and volatility, which may serve to hinder the dissemintation of TTDR-HSV. The challenge for future studies would be to explore how TTDRHSV could be redesigned and/or employed with combinatorial approaches to better target and destroy the heterogeneous and dynamic cell populations in the aggressive tumor mass.

Transcriptional and translational dual-regulated oncolytic herpes simplex virus type 1 for targeting prostate tumors.

The aim of this project was to demonstrate that an oncolytic herpes simplex virus type 1 (HSV-1) can replicate in a tissue- and tumor-specific fashion through both transcriptional (prostate-specific promoter, ARR(2)PB) and translational (5'-untranslated regions (5'UTRs) of rFGF-2) regulation of an essential viral gene, ICP27. We generated two recombinant viruses, ARR(2)PB-ICP27 (A27) and ARR(2)PB-5'UTR-ICP27 (AU27) and tested their efficacy and toxicity both in vitro and in vivo. The ARR(2)PB promoter caused overexpression of ICP27 gene in the presence of activated androgen receptors (ARs) and increased viral replication in prostate cells. However, this transcriptional upregulation was effectively constrained by the 5'UTR-mediated translational regulation. Mice bearing human prostate LNCaP tumors, treated with a single intravenous injection of 5 x 10(7) plaque-forming units (pfu) of AU27 virus exhibited a >85% reduction in tumor size at day 28 after viral injection. Although active viral replication was readily evident in the tumors, no viral DNA was detectable in normal organs as measured by real-time PCR analyses. In conclusion, a transcriptional and translational dual-regulated (TTDR) viral essential gene expression can increase both viral lytic activity and tumor specificity, and this provides a basis for the development of a novel tumor-specific oncolytic virus for systemic treatment of locally advanced and metastatic prostate cancers.

Down-regulation of type I interferon receptor sensitizes bladder cancer cells to vesicular stomatitis virus-induced cell death.

The intrinsic oncolytic specificity of vesicular stomatitis virus (VSV) is currently being exploited to develop alternative therapeutic strategies for bladder cancer and other cancers. Previously we reported that oncolytic VSV is a potent agent for intravesical treatment of high risk bladder cancer. We observed that VSV preferentially targeted bladder cancer cells resistant to type I interferon (IFN) treatment. The goal of the current study was to further elucidate the nature of the molecular defect of IFN signaling by which bladder cancer cells become susceptible to VSV infection. Using a tissue microarray composed of human bladder cancer cores, we observed that expression of type I IFN receptor (IFNAR) was decreased relative to normal bladder tissue. Advanced bladder cancers had even lower expression of IFNAR. We found that bladder cancer cells susceptible to VSV-induced lysis had low expression of IFNAR as well. We hypothesized that down-regulation of IFNAR in bladder cancer cells may be a molecular mechanism responsible for resistance to type I IFN treatment and sensitivity to VSV oncolysis. SiRNA knockdown of IFNAR indeed facilitated replication of VSV in cells previously resistant to VSV treatment. Blocking IFNAR with a neutralizing antibody showed a similar effect. Hence down-regulation of IFNAR in bladder cancer may be one of the primary molecular mechanisms for clinical IFN resistance. However, this also facilitates VSV replication and oncolysis in high risk bladder cancers and provides a basis for selecting bladder cancer patients for IFN or oncolytic VSV therapy in future clinical trials.

MicroRNA regulation of oncolytic herpes simplex virus-1 for selective killing of prostate cancer cells.

PURPOSE: Advanced castration-resistant prostate cancer, for which there are few treatment options, remains one of the leading causes of cancer death. MicroRNAs (miRNA) have provided a new opportunity for more stringent regulation of tumor-specific viral replication. The purpose of this study was to provide a proof-of-principle that miRNA-regulated oncolytic herpes simplex virus-1 (HSV-1) virus can selectively target cancer cells with reduced toxicity to normal tissues. EXPERIMENTAL DESIGN: We incorporated multiple copies of miRNA complementary target sequences (for miR-143 or miR-145) into the 3'-untranslated region (3'-UTR) of an HSV-1 essential viral gene, ICP4, to create CMV-ICP4-143T and CMV-ICP4-145T amplicon viruses and tested their targeting specificity and efficacy both in vitro and in vivo. RESULTS: Although miR-143 and miR-145 are highly expressed in normal tissues, they are significantly down-regulated in prostate cancer cells. We further showed that miR-143 and miR-145 inhibited the expression of the ICP4 gene at the translational level by targeting the corresponding 3'-UTR in a dose-dependent manner. This enabled selective viral replication in prostate cancer cells. When mice bearing LNCaP human prostate tumors were treated with these miRNA-regulated oncolytic viruses, a >80% reduction in tumor volume was observed, with significantly attenuated virulence to normal tissues in comparison with control amplicon viruses not carrying these 3'-UTR sequences. CONCLUSION: Our study is the first to show that inclusion of specific miRNA target sequences into the 3'-UTR of an essential HSV-1 gene is a viable strategy for restricting viral replication and oncolysis to cancer cells while sparing normal tissues.

Cell death in the Purkinje cells of the cerebellum of senescence accelerated mouse (SAMP(8)).

The cerebella of SAMP(8) (accelerated aging mouse) and SAMR(1) controls were analyzed by Western Blotting of tyrosine hydroxylase and choline acetyltransferase, as well as by TUNEL and histological silver staining. Both tyrosine hydroxylase and choline acetyltransferase levels were higher in SAMR(1) than in SAMP(8). There was also an age-related decrease in enzyme levels in SAMP(8), with the reduction of tyrosine hydroxylase being more apparent. Concomitantly, there was an age-related increase of apoptosis in the medial neocerebellum and the vermis as revealed by TUNEL, with changes being significant in the SAMP(8) strain. Histologically, some Purkinje cells appeared to disappear during aging. Taken together, the data suggests that the aging SAMP(8) strain displays differential Purkinje cell death in the medial cerebellum and that some of the dying cells are likely to be catecholaminergic.

The SRS superfamily of Toxoplasma surface proteins.

The surface of the protozoan parasite Toxoplasma gondii is coated with developmentally expressed, glycosylphosphatidylinositol-linked proteins structurally related to the highly immunogenic surface antigen SAG1. Collectively, these surface antigens are known as the SRS (SAG1-related sequences) superfamily of proteins. SRS proteins are thought to mediate attachment to host cells and activate host immunity to regulate the parasite's virulence. To better understand the number, evolution and developmental expression of SRS genes, this study has bioinformatically identified 161 unique SRS DNA sequences present in the T. gondii type II Me49 genome. The SRS superfamily of sequences phylogenetically bifurcates into two subfamilies, the prototypic members being SAG1 and SAG2A, respectively. Paralogous SRS sequences are 24-99% identical, are tandemly arrayed throughout the genome, and are present on most, if not all, chromosomes. All 11 SRS sequences on chromosomes Ia and Ib are clustered at sub-telomeric expression sites. Messenger RNA expression in the majority of SRS sequences for which multiple Expressed Sequence Tags exist is developmentally regulated. A consensus nucleotide sequence surrounding both the splice acceptor and donor sites was identified in those SRS sequences possessing an intron. Genotypic differences among SRS sequences are present at several loci (e.g. the absence of SAG5B, the truncation of SAG2D in Me49 compared with RH) indicating that different genotypes possess distinct sets of SRS sequences. Orthologous genes are restricted to tissue-dwelling coccidia (Neospora, Sarcocystis) with no related sequences present in other more distant apicomplexa such as Eimeria, Cryptosporidia, and Plasmodium spp.