Vascular mimicry: Triggers, molecular interactions and in vivo models.

Vascular mimicry is induced by a wide array of genes with functions related to cancer stemness, hypoxia, angiogenesis and autophagy. Vascular mimicry competent (VM-competent) cells that form de novo blood vessels are common in solid tumors facilitating tumor cell survival and metastasis. VM-competent cells display increased levels of vascular mimicry selecting for stem-like cells in an O2-gradient-dependent manner in deeply hypoxic tumor regions, while also aiding in maintaining tumor cell metabolism and stemness. Three of the principal drivers of vascular mimicry are EphA2, Nodal and HIF-1α, however, directly or indirectly many of these molecules affect VE-Cadherin (VE-Cad), which forms gap-junctions to bind angiogenic blood vessels together. During vascular mimicry, the endothelial-like functions of VM-competent cancer stem cells co-opt VE-Cad to bind cancer cells together to create cancer cell-derived blood conducting vessels. This process potentially compensates for the lack of access to blood and nutrient in avascular tumors, simultaneously providing nutrients and enhancing cancer invasion and metastasis. Current evidence also supports that vascular mimicry promotes cancer malignancy and metastasis due to the cooperation of oncogenic signaling molecules driving cancer stemness and autophagy. While a number of currently used cancer therapeutics are effective inhibitors of vascular mimicry, developing a new class of vascular mimicry specific inhibitors could allow for the treatment of angiogenesis-resistant tumors, inhibit cancer metastasis and improve patient survival. In this review, we describe the principal vascular mimicry pathways in addition to emphasizing the roles of hypoxia, autophagy and select proangiogenic oncogenes in this process.

MDA-9/Syntenin (SDCBP): Novel gene and therapeutic target for cancer metastasis.

The primary cause of cancer-related death from solid tumors is metastasis. While unraveling the mechanisms of this complicated process continues, our ability to effectively target and treat it to decrease patient morbidity and mortality remains disappointing. Early detection of metastatic lesions and approaches to treat metastases (both pharmacological and genetic) are of prime importance to obstruct this process clinically. Metastasis is complex involving both genetic and epigenetic changes in the constantly evolving tumor cell. Moreover, many discrete steps have been identified in metastatic spread, including invasion, intravasation, angiogenesis, attachment at a distant site (secondary seeding), extravasation and micrometastasis and tumor dormancy development. Here, we provide an overview of the metastatic process and highlight a unique pro-metastatic gene, melanoma differentiation associated gene-9/Syntenin (MDA-9/Syntenin) also called syndecan binding protein (SDCBP), which is a major contributor to the majority of independent metastatic events. MDA-9 expression is elevated in a wide range of carcinomas and other cancers, including melanoma, glioblastoma multiforme and neuroblastoma, suggesting that it may provide an appropriate target to intervene in metastasis. Pre-clinical studies confirm that inhibiting MDA-9 either genetically or pharmacologically profoundly suppresses metastasis. An additional benefit to blocking MDA-9 in metastatic cells is sensitization of these cells to a second therapeutic agent, which converts anti-invasion effects to tumor cytocidal effects. Continued mechanistic and therapeutic insights hold promise to advance development of truly effective therapies for metastasis in the future.

The role of JNK phosphorylation as a molecular target to enhance adenovirus replication, oncolysis and cancer therapeutic efficacy.

Oncolytic adenoviruses (Ads) are cancer selective tumoricidal agents; however their mechanism of Ad-mediated cancer cell lysis, or oncolysis, remains undefined. This report focuses upon the autophagy mediator c-JUN n-terminal kinase (JNK) and its effects upon Ad oncolysis and replication. Previously, E1b-deleted Ads have been used to treat several hundred cancer patients with limited clinical efficacy. We hypothesize that by studying the potential interactions between E1b and JNK, mechanisms to improve oncolytic Ad design and cancer therapeutic efficacy may be elucidated. To test this hypothesis, E1b was selectively deleted from the Ad genome. These studies indicated that Ads encoding E1b induced JNK phosphorylation predominately occurred via E1b-19K. The expression of another crucial Ad gene E1a was then overexpressed by the CMV promoter via the replication competent Ad vector Adhz69; these data indicated that E1A also induced JNK phosphorylation. To assess the effects of host cell JNK expression upon Ad oncolysis and replication, siRNA targeting JNK1 and JNK2 (JNK1/2) were utilized. The oncolysis and replication of the E1b-19K wild-type Ads Ad5 and Adhz63 were significantly attenuated following JNK1/2 siRNA transfection. However the oncolytic effects and replication of the E1b-19K deleted Ad Adhz60 were not altered by JNK1/2 siRNA transfection, further implicating the crucial role of E1b-19K for Ad oncolysis and replication via JNK phosphorylation. This study has demonstrated for the first time that JNK is an intriguing molecular marker associated with enhanced Ad virotherapy efficacy, influencing future Ad vector design.

Prospects of Gene Therapy to Treat Melanoma.

The incidence of melanoma has continued to increase over the past 30 years. Hence, developing effective therapies to treat both primary and metastatic melanoma are essential. While advances in targeted therapy and immunotherapy have provided novel therapeutic options to treat melanoma, gene therapy may provide additional strategies for the treatment of metastatic melanoma clinically. This review focuses upon the challenges and opportunities that gene therapy provides for targeting melanoma. We begin with a discussion of the various gene therapy targets which are relevant to melanoma. Next, we explore the gene therapy clinical trials that have been conducted for treating melanoma. Finally, challenges faced in gene therapy as well as combination therapies for targeting melanoma, which may circumvent these obstacles, will be discussed. Targeted combination gene therapy strategies hold significant promise for developing the most effective therapeutic outcomes, while reducing the toxicity to noncancerous cells, and would integrate the patient's immune system to diminish melanoma progression. Next-generation vectors designed to embody required safety profiles and "theranostic" attributes, combined with immunotherapeutic strategies would be critical in achieving beneficial management and therapeutic outcomes in melanoma patients.

New Insights Into Beclin-1: Evolution and Pan-Malignancy Inhibitor Activity.

Autophagy is a functionally conserved self-degradation process that facilitates the survival of eukaryotic life via the management of cellular bioenergetics and maintenance of the fidelity of genomic DNA. The first known autophagy inducer was Beclin-1. Beclin-1 is expressed in multicellular eukaryotes ranging throughout plants to animals, comprising a nonmonophyllic group, as shown in this report via aggressive BLAST searches. In humans, Beclin-1 is a haploinsuffient tumor suppressor as biallelic deletions have not been observed in patient tumors clinically. Therefore, Beclin-1 fails the Knudson hypothesis, implicating expression of at least one Beclin-1 allele is essential for cancer cell survival. However, Beclin-1 is frequently monoallelically deleted in advanced human cancers and the expression of two Beclin-1 allelles is associated with greater anticancer effects. Overall, experimental evidence suggests that Beclin-1 inhibits tumor formation, angiogenesis, and metastasis alone and in cooperation with the tumor suppressive molecules UVRAG, Bif-1, Ambra1, and MDA-7/IL-24 via diverse mechanisms of action. Conversely, Beclin-1 is upregulated in cancer stem cells (CSCs), portending a role in cancer recurrence, and highlighting this molecule as an intriguing molecular target for the treatment of CSCs. Many aspects of Beclin-1's biological effects remain to be studied. The consequences of these BLAST searches on the molecular evolution of Beclin-1, and the eukaryotic branches of the tree of life, are discussed here in greater detail with future inquiry focused upon protist taxa. Also in this review, the effects of Beclin-1 on tumor suppression and cancer malignancy are discussed. Beclin-1 holds significant promise for the development of novel targeted cancer therapeutics and is anticipated to lead to a many advances in our understanding of eukaryotic evolution, multicellularity, and even the treatment of CSCs in the coming decades.

Adenovirus Lacking E1b Efficiently Induces Cytopathic Effect in HPV-16-Positive Murine Cancer Cells via Virus Replication and Apoptosis.

Conditionally replicative adenoviruses (CRAds) replicate poorly in murine cancer cells; however, E1b-deleted CRAds may replicate effectively in HPV16-E6/E7-positive murine cancer cells (TC-1). The HPV16 E7 open reading frame encodes functions analogous to these deleted adenovirus E1 proteins. In this study, an E1b-deleted CRAd (Adhz60) was evaluated for its ability to replicate and induce oncolysis in TC-1 cells. Adhz60-mediated oncolysis was similar in TC-1 and HeLa cells. Productive viral replication was evident based on expression of E1A and hexon, production of infectious virus progeny, and Adhz60-induced apoptosis. The results suggest that TC-1 murine cancer cells allow Adhz60 replication and oncolysis.

Adenovirus with DNA Packaging Gene Mutations Increased Virus Release.

Adenoviruses (Ads) have been extensively manipulated for the development of cancer selective replication, leading to cancer cell death or oncolysis. Clinical studies using E1-modified oncolytic Ads have shown that this therapeutic platform was safe, but with limited efficacy, indicating the necessity of targeting other viral genes for manipulation. To improve the therapeutic efficacy of oncolytic Ads, we treated the entire Ad genome repeatedly with UV-light and have isolated AdUV which efficiently lyses cancer cells as reported previously (Wechman, S. L. et al. Development of an Oncolytic Adenovirus with Enhanced Spread Ability through Repeated UV Irradiation and Cancer Selection. Viruses2016, 8, 6). In this report, we show that no mutations were observed in the early genes (E1 or E4) of AdUV while several mutations were observed within the Ad late genes which have structural or viral DNA packaging functions. This study also reported the increased release of AdUV from cancer cells. In this study, we found that AdUV inhibits tumor growth following intratumoral injection. These results indicate the potentially significant role of the viral late genes, in particular the DNA packaging genes, to enhance Ad oncolysis.

Development of an Oncolytic Adenovirus with Enhanced Spread Ability through Repeated UV Irradiation and Cancer Selection.

Oncolytic adenoviruses (Ads) have been shown to be safe and have great potential for the treatment of solid tumors. However, the therapeutic efficacy of Ads is antagonized by limited spread within solid tumors. To develop Ads with enhanced spread, viral particles of an E1-wildtype Ad5 dl309 was repeatedly treated with UV type C irradiation and selected for the efficient replication and release from cancer cells. After 72 cycles of treatment and cancer selection, AdUV was isolated. This vector has displayed many favorable characteristics for oncolytic therapy. AdUV was shown to lyse cancer cells more effectively than both E1-deleted and E1-wildtype Ads. This enhanced cancer cell lysis appeared to be related to increased AdUV replication in and release from infected cancer cells. AdUV-treated A549 cells displayed greater expression of the autophagy marker LC3-II during oncolysis and formed larger viral plaques upon cancer cell monolayers, indicating increased virus spread among cancer cells. This study indicates the potential of this approach of irradiation of entire viral particles for the development of oncolytic viruses with designated therapeutic properties.

Combined therapy of oncolytic adenovirus and temozolomide enhances lung cancer virotherapy in vitro and in vivo.

Oncolytic adenoviruses (OAds) are very promising for the treatment of lung cancer. However, OAd-based monotherapeutics have not been effective during clinical trials. Therefore, the effectiveness of virotherapy must be enhanced by combining OAds with other therapies. In this study, the therapeutic potential of OAd in combination with temozolomide (TMZ) was evaluated in lung cancer cells in vitro and in vivo. The combination of OAd and TMZ therapy synergistically enhanced cancer cell death; this enhanced cancer cell death may be explained via three related mechanisms: apoptosis, virus replication, and autophagy. Autophagy inhibition partially protected cancer cells from this combined therapy. This combination significantly suppressed the growth of subcutaneous H441 lung cancer xenograft tumors in athymic nude mice. In this study, we have provided an experimental rationale to test OAds in combination with TMZ in a lung cancer clinical trial.

Oncolytic Replication of E1b-Deleted Adenoviruses.

Various viruses have been studied and developed for oncolytic virotherapies. In virotherapy, a relatively small amount of viruses used in an intratumoral injection preferentially replicate in and lyse cancer cells, leading to the release of amplified viral particles that spread the infection to the surrounding tumor cells and reduce the tumor mass. Adenoviruses (Ads) are most commonly used for oncolytic virotherapy due to their infection efficacy, high titer production, safety, easy genetic modification, and well-studied replication characteristics. Ads with deletion of E1b55K preferentially replicate in and destroy cancer cells and have been used in multiple clinical trials. H101, one of the E1b55K-deleted Ads, has been used for the treatment of late-stage cancers as the first approved virotherapy agent. However, the mechanism of selective replication of E1b-deleted Ads in cancer cells is still not well characterized. This review will focus on three potential molecular mechanisms of oncolytic replication of E1b55K-deleted Ads. These mechanisms are based upon the functions of the viral E1B55K protein that are associated with p53 inhibition, late viralmRNAexport, and cell cycle disruption.

Oncolytic adenovirus targeting cyclin E overexpression repressed tumor growth in syngeneic immunocompetent mice.

BACKGROUND: Clinical trials have indicated that preclinical results obtained with human tumor xenografts in mouse models may overstate the potential of adenovirus (Ad)-mediated oncolytic therapies. We have previously demonstrated that the replication of human Ads depends on cyclin E dysregulation or overexpression in cancer cells. ED-1 cell derived from mouse lung adenocarcinomas triggered by transgenic overexpression of human cyclin E may be applied to investigate the antitumor efficacy of oncolytic Ads. METHODS: Ad-cycE was used to target cyclin E overexpression in ED-1 cells and repress tumor growth in a syngeneic mouse model for investigation of oncolytic virotherapies. RESULTS: Murine ED-1 cells were permissive for human Ad replication and Ad-cycE repressed ED-1 tumor growth in immunocompetent FVB mice. ED-1 cells destroyed by oncolytic Ads in tumors were encircled in capsule-like structures, while cells outside the capsules were not infected and survived the treatment. CONCLUSION: Ad-cycE can target cyclin E overexpression in cancer cells and repress tumor growth in syngeneic mouse models. The capsule structures formed after Ad intratumoral injection may prevent viral particles from spreading to the entire tumor.