Dawn E. Post

PTEN and Hypoxia Regulate Tissue Factor Expression and Plasma Coagulation by Glioblastoma

We have previously proposed that intravascular thrombosis and subsequent vasoocclusion contribute to the development of pseudopalisading necrosis, a pathologic hallmark that distinguishes glioblastoma (WHO grade 4) from lower grade astrocytomas. To better understand the potential prothrombotic mechanisms underlying the formation of these structures that drive tumor angiogenesis, we investigated tissue factor (TF), a potent procoagulant protein known to be overexpressed in astrocytomas. We hypothesized that PTEN loss and tumor hypoxia, which characterize glioblastoma but not lower grade astrocytomas, could up-regulate TF expression and cause intravascular thrombotic occlusion. We examined the effect of PTEN restoration and hypoxia on TF expression and plasma coagulation using a human glioma cell line containing an inducible wt-PTEN cDNA. Cell exposure to hypoxia (1% O(2)) markedly increased TF expression, whereas restoration of wt-PTEN caused decreased cellular TF. The latter effect was at least partially dependent on PTENs protein phosphatase activity. Hypoxic cells accelerated plasma clotting in tilt tube assays and this effect was prevented by both inhibitory antibodies to TF and plasma lacking factor VII, implicating TF-dependent mechanisms. To further examine the genetic events leading to TF up-regulation during progression of astrocytomas, we investigated its expression in a series of human astrocytes sequentially infected with E6/E7/human telomerase, Ras, and Akt. Cells transformed with Akt showed the greatest incremental increase in hypoxia-induced TF expression and secretion. Together, our results show that PTEN loss and hypoxia up-regulate TF expression and promote plasma clotting by glioma cells, suggesting that these mechanisms may underlie intravascular thrombosis and pseudopalisading necrosis in glioblastoma.

Use of Replicating Oncolytic Adenoviruses in Combination Therapy for Cancer

Oncolytic virotherapy is the use of genetically engineered viruses that specifically target and destroy tumor cells via their cytolytic replication cycle. Viral-mediated tumor destruction is propagated through infection of nearby tumor cells by the newly released progeny. Each cycle should amplify the number of oncolytic viruses available for infection. Our understanding of the life cycles of cytolytic viruses has allowed manipulation of their genome to selectively kill tumor cells over normal tissue. Because the mechanism of tumor destruction is different, oncolytic virotherapy should work synergistically with current modes of treatment such as chemotherapy and radiation therapy. This article focuses on oncolytic adenoviruses that have been created and tested in preclinical and clinical trials in combination with chemotherapy, radiation therapy, and gene therapy.

A novel hypoxia-inducible factor (HIF)activated oncolytic adenovirus for cancer therapy

New therapy targeting the hypoxic fraction of tumors needs to be designed as this population of cells is the most resistant to radio- and chemotherapies. Hypoxia-inducible factor (HIF) mediates transcriptional responses to hypoxia by binding to hypoxia-responsive elements (HRE) in target genes. We developed a hypoxia/HIF-dependent replicative adenovirus (HYPR-Ad) to target hypoxic cells. HYPR-Ad displays hypoxia-dependent E1A expression and conditional cytolysis of hypoxic but not normoxic cells. This work provides proof-of-principle evidence that an attenuated oncolytic adenovirus that selectively lyses cells under hypoxia can be generated. This therapeutic approach can be used to treat all solid tumors that develop hypoxia, regardless of their tissue origin or genetic alterations.

Generation of bidirectional hypoxia/HIF-responsive expression vectors to target gene expression to hypoxic cells

Hypoxia initiates an adaptive physiological response in all organisms and plays a role in the pathogenesis of several human diseases. The hypoxia/HIF-inducible factor-1 (HIF-1) transcription factor mediates transcriptional responses to hypoxia by binding to a cis-acting hypoxia-responsive element (HRE) present within target genes. The use of the HIF-1/HRE system of gene regulation can be utilized as a mechanism to target expression of therapeutic genes to hypoxic cells or cells that have a constitutively active HIF-1/HRE pathway due to cell transformation. Given the rapid resistance of tumors to single therapeutic strategies, new vector systems need to be developed that can deliver multimodal therapy. Here we show that HREs function as classical enhancer elements and function bidirectionally to co-regulate the expression of two genes. We designed a large series of novel bidirectional hypoxia/HIF-responsive expression vectors using HREs derived from the human vascular endothelial growth factor (VEGF) and erythropoietin (EPO) genes. We measured the ability of these constructs to express the luciferase and LacZ/β-galactosidase (β-gal) reporter genes bidirectionally under normoxic (21% O2) versus hypoxic (1, 3, 5, and 10% O2) conditions by transient transfection in three human glioma cell lines (LN229, U251MG and U138MG). Nine constructs were identified that exhibited moderate to high inducibility at 1% O2 while maintaining tight regulation under normoxic conditions. Moreover, the level of activation was a function of O2 concentration and was exponential at O2 levels below 5%. These vectors will be valuable tools in a variety of gene therapy applications targeting pathological activation of the HIF-1/HRE pathway.

Tumor-specific gene expression using the survivin promoter is further increased by hypoxia

Increasing evidence indicates that survivin, an inhibitor of apoptosis protein (IAP), is expressed in human cancer cells but is absent from most normal adult tissues. Here, we examined the feasibility of using a survivin promoter (Sur-P) to direct therapeutic expression of a proapoptotic gene specifically in human tumor cells. First, we demonstrated that this promoter was highly active in human tumor cells but not in normal cells. Second, we found that Sur-P activity was upregulated by hypoxia in tumor cells. Third, to further enhance this promoters activity under hypoxia, we added a hypoxia-responsive element (HRE) from the vascular endothelial growth factor gene promoter in its 5′ region, and showed that this combination resulted in a further increase in the level of gene expression in hypoxic tumor cells. Finally, we demonstrated that expression of an autocatalytic reverse caspase-3 gene by this promoter specifically induced apoptotic cell death in human tumor cells but not in normal cells. These findings support the use of promoters Sur-P or chimeric HRE-Sur-P for generating novel vectors for cancer gene therapy.

Targeted Cancer Gene Therapy Using a Hypoxia Inducible Factor–Dependent Oncolytic Adenovirus Armed with Interleukin-4

There is a need for novel therapies targeting hypoxic cells in tumors. These cells are associated with tumor resistance to therapy and express hypoxia inducible factor-1 (HIF-1), a transcription factor that mediates metabolic adaptation to hypoxia and activates tumor angiogenesis. We previously developed an oncolytic adenovirus (HYPR-Ad) for the specific killing of hypoxic/HIF-active tumor cells, which we now armed with an interleukin-4 gene (HYPR-Ad-IL4). We designed HYPR-Ad-IL4 by cloning the Ad E1A viral replication and IL-4 genes under the regulation of a bidirectional hypoxia/HIF-responsive promoter. The IL-4 cytokine was chosen for its ability to induce a strong host antitumor immune response and its potential antiangiogenic activity. HYPR-Ad-IL4 induced hypoxia-dependent IL-4 expression, viral replication, and conditional cytolysis of hypoxic, but not normoxic cells. The treatment of established human tumor xenografts with HYPR-Ad-IL4 resulted in rapid and maintained tumor regression with the same potency as that of wild-type dl309-Ad. HYPR-Ad-IL4-treated tumors displayed extensive necrosis, fibrosis, and widespread viral replication. Additionally, these tumors contained a distinctive leukocyte infiltrate and prominent hypoxia. The use of an oncolytic Ad that locally delivers IL-4 to tumors is novel, and we expect that HYPR-Ad-IL4 will have broad therapeutic use for all solid tumors that have hypoxia or active HIF, regardless of tissue origin or genetic alterations.

Replicative oncolytic adenoviruses in multimodal cancer regimens.

The use of replication-competent viruses that have a cytolytic cycle has emerged as a viable strategy (oncolytic virotherapy) to specifically kill tumor cells and the field has advanced to the point of clinical trials. A theoretical advantage of replicative oncolytic viruses is that their numbers should increase via viral replication within infected tumor cells and resulting viral progeny can then infect additional cells within the tumor mass. The life cycle of a virus involves multiple interactions between viral and cellular proteins/genes, which maximize the ability of the virus to infect and replicate within cells. Understanding such interactions has led to the design of numerous genetically engineered adenovirus (Ad) vectors that selectively kill tumor cells while sparing normal cells. These viruses have also been modified to function as therapeutic gene delivery vehicles, thus augmenting their anticancer capacity. In addition, the oncolytic mode of tumor killing differs from that of standard anticancer therapies, providing the possibility for synergistic interactions with other therapies in a multimodal antitumor approach. In this review, we describe the oncolytic Ad vectors tested in preclinical and clinical models and their use in combination with chemo-, radio-, and gene therapies.

Cancer Therapy with a Replicating Oncolytic Adenovirus Targeting the Hypoxic Microenvironment of Tumors

Hypoxia plays a critical role in driving tumor malignancy and is associated with poor patient survival in many human cancers. Novel therapies targeting hypoxic tumor cells are urgently needed, because these cells hinder tumor eradication. Here we demonstrate than an anticancer strategy based on intratumoral delivery of a novel type of oncolytic adenovirus targeting tumor hypoxia is therapeutically efficient and can augment standard chemotherapy. We used a conditionally replicative adenovirus (HYPR-Ad) to specifically kill hypoxic tumor cells. Viral infection and conditional replication occurred efficiently in hypoxic/hypoxia-inducible factor-active cells in culture and in vivo, prevented tumor formation, and reduced the growth of established tumors. Combining HYPR-Ad with chemotherapy effective against normoxic cells resulted in strongly enhanced antitumor efficacy. These studies demonstrate that targeting the hypoxic microenvironment of tumors rather than an intrinsic gene expression defect is a viable and novel antitumor therapeutic strategy that can be used in combination with existing treatment regimens. The replication and oncolytic potential of this virus was made dependent on hypoxic/hypoxia-inducible factor, a transcription factor activated in the tumor hypoxic microenvironment, broadening its therapeutic use to solid tumors of any genetic make-up or tissue of origin.

Replicative oncolytic herpes simplex viruses in combination cancer therapies.

Viruses that kill the host cell during their replication cycle have attracted much interest for the specific killing of tumor cells and this oncolytic virotherapy is being evaluated in clinical trials. The rationale for using replicative oncolytic viruses is that viral replication in infected tumor cells will permit in situ viral multiplication and spread of viral infection throughout the tumor mass thus overcoming the delivery problems of gene therapy. Improved understanding of the life cycle of viruses has evidenced multiple interactions between viral and cellular gene products, which have evolved to maximize the ability of viruses to infect and multiply within cells. Differences in viral-cell interactions between normal and tumor cells have emerged that have led to the design of a number of genetically engineered viral vectors that selectively kill tumor cells while sparing normal cells. These viruses have undergone further modifications to carry adjunct therapy genes to increase their anti-cancer abilities. Since these viruses kill cells by oncolytic mechanisms differing from standard anticancer therapies, there is an opportunity that synergistic interactions with other therapies might be found with the use of combination therapy. In this review, we focus on the oncolytic Herpes Simplex Virus-1 (HSV-1) vectors that have been examined in preclinical and clinical cancer models and their use in combination with chemo-, radio-, and gene therapies.

Epidermal Growth Factor Receptor as a Therapeutic Target in Glioblastoma

Glioblastoma represents one of the most challenging problems in neurooncology. Among key elements driving its behavior is the transmembrane epidermal growth factor receptor family, with the first member epidermal growth factor receptor (EGFR) centered in most studies. Engagement of the extracellular domain with a ligand activates the intracellular tyrosine kinase (TK) domain of EGFR, leading to autophosphorylation and signal transduction that controls proliferation, gene transcription, and apoptosis. Oncogenic missense mutations, deletions, and insertions in the EGFR gene are preferentially located in the extracellular domain in glioblastoma and cause constitutive activation of the receptor. The mutant EGFR may also transactivate other cell surface molecules, such as additional members of the EGFR family and the platelet-derived growth factor receptor, which ignite signaling cascades that synergize with the EGFR-initiated cascade. Because of the cell surface location and increased expression of the receptor along with its important biological function, EGFR has triggered much effort for designing targeted therapy. These approaches include TK inhibition, monoclonal antibody, vaccine, and RNA-based downregulation of the receptor. Treatment success requires that the drug penetrates the blood–brain barrier and has low systemic toxicity but high selectivity for the tumor. While the blockade of EGFR-dependent processes resulted in experimental and clinical treatment success, cells capable of using alternative signaling ultimately escape this strategy. A combination of interventions targeting tumor-specific cell surface regulators along with convergent downstream signaling pathways will likely enhance efficacy. Studies on EGFR in glioblastoma have revealed much information about the complexity of gliomagenesis and also facilitated the development of strategies for targeting drivers of tumor growth and combination therapies with increasing complexity.