Vesicular stomatitis virus (VSV) is an oncolytic virus that induces cancer cell death through activation of the apoptotic pathway, although intrinsic resistance to oncolysis is found in some cell lines and many primary tumors, as a consequence of residual innate immunity to VSV. In resistant tumor models, VSV oncolytic potential can be reversibly stimulated by combination with epigenetic modulators such as the histone deacetylase inhibitor Vorinostat. Based on this reversible effect of Vorinostat, we reasoned that critical host genes involved in oncolysis may likewise be reversibly regulated by Vorinostat. A transcriptome analysis in prostate cancer PC3 cells identified a subset of NF-?B target genes reversibly regulated by Vorinostat, as well as a group of interferon (IFN)-stimulated genes (ISGs). Consistent with the induction of NF-?B target genes, Vorinostat-mediated enhancement of VSV oncolysis increased hyper-acetylation of NF-?B RELA/p65. Additional bioinformatics analysis revealed that NF-?B signaling also increased expression of several autophagy-related genes. Kinetically, autophagy preceded apoptosis and apoptosis was only observed when cells were treated with both VSV and Vorinostat. VSV replication and cell killing were suppressed when NF-?B signaling was inhibited using pharmacological or genetic approaches. Inhibition of autophagy by 3-methyladenine (3-MA) enhanced expression of ISGs, and either 3-MA treatment or genetic ablation of the autophagic marker Atg5 decreased VSV replication and oncolysis. Together, these data demonstrate that Vorinostat stimulates NF-?B activity in a reversible manner via modulation of RELA/p65 signaling, leading to induction of autophagy, suppression of the IFN-mediated response, and subsequent enhancement of VSV replication and apoptosis.
Many primary cancers including chronic lymphocytic leukemia (CLL) are resistant to vesicular stomatitis virus (VSV)-induced oncolysis due to overexpression of the antiapoptotic and antiautophagic members of the B-cell lymphoma-2 (BCL-2) family. In the present study, we investigated the mechanisms of CLL cell death induced as a consequence of VSV infection in the presence of BCL-2 inhibitors, obatoclax, and ABT-737 in primary ex vivo CLL patient samples. Microarray analysis of primary CD19? CD5? CLL cells treated with obatoclax and VSV revealed changes in expression of genes regulating apoptosis, the mechanistic target of rapamycin (mTOR) pathway, and cellular metabolism. A combined therapeutic effect was observed for VSV and BCL-2 inhibitors in cells from untreated patients and from patients unresponsive to standard of care therapy. In addition, combination treatment induced several markers of autophagy--LC3-II accumulation, p62 degradation, and staining of autophagic vacuoles. Inhibition of early stage autophagy using 3-methyladenine (3-MA) led to increased apoptosis in CLL samples. Mechanistically, a combination of BCL-2 inhibitors and VSV disrupted inhibitory interactions of Beclin-1 with BCL-2 and myeloid cell leukemia-1 (MCL-1), thus biasing cells toward autophagy. We propose a mechanism in which changes in cellular metabolism, coupled with pharmacologic disruption of the BCL-2-Beclin-1 interactions, facilitate induction of apoptosis and autophagy to mediate the cytolytic effect of VSV.
The balance between the pro-apoptotic lipids ceramide and sphingosine and the pro-survival lipid sphingosine 1-phosphate (S1P) is termed the "sphingosine rheostat". Two isozymes, sphingosine kinase 1 and 2 (SK1 and SK2), are responsible for phosphorylation of pro-apoptotic sphingosine to form pro-survival S1P. We have previously reported the antitumor properties of an SK2 selective inhibitor, ABC294640, alone or in combination with the multikinase inhibitor sorafenib in mouse models of kidney carcinoma and pancreatic adenocarcinoma. Here we evaluated the combined antitumor effects of the aforementioned drug combination in two mouse models of hepatocellular carcinoma. Although combining the SK2 inhibitor, ABC294640, and sorafenib in vitro only afforded additive drug-drug effects, their combined antitumor properties in the mouse model bearing HepG2 cells mirrored effects previously observed in animals bearing kidney carcinoma and pancreatic adenocarcinoma cells. Combining ABC294640 and sorafenib led to a decrease in the levels of phosphorylated ERK in SK-HEP-1 cells, indicating that the antitumor effect of this drug combination is likely mediated through a suppression of the MAPK pathway in hepatocellular models. We also measured levels of S1P in the plasma of mice treated with two different doses of ABC294640 and sorafenib. We found decreases in the levels of S1P in plasma of mice treated daily with 100 mg/kg of ABC294640 for 5 weeks, and this decrease was not affected by co-administration of sorafenib. Taken together, these data support combining ABC294640 and sorafenib in clinical trials in HCC patients. Furthermore, monitoring levels of S1P may provide a pharmacodynamic marker of ABC294640 activity.
The pro-apoptotic lipid sphingosine is phosphorylated by sphingosine kinases 1 and 2 (SK1 and SK2) to generate the mitogenic lipid sphingosine-1-phosphate (S1P). We previously reported that inhibition of SK activity delays tumor growth in a mouse mammary adenocarcinoma model. Because SK inhibitors and the multikinase inhibitor sorafenib both suppress the MAP kinase pathway, we hypothesized that their combination may provide enhanced inhibition of tumor growth. Therefore, we evaluated the effects of two SK inhibitors, ABC294640 (a SK2-specific inhibitor) and ABC294735 (a dual SK1/SK2 inhibitor), alone and in combination with sorafenib on human pancreatic adenocarcinoma (Bxpc-3) and kidney carcinoma (A-498) cells in vitro and in vivo. Exposure of either Bxpc-3 or A-498 cells to combinations of ABC294640 and sorafenib or ABC294735 and sorafenib resulted in synergistic cytotoxicity, associated with activation of caspases 3/7 and DNA fragmentation. Additionally, strong decreases in ERK phosphorylation were observed in Bxpc-3 and A-498 cells exposed to either the sorafenib/ABC294640 or the sorafenib/ABC294735 combination. Oral administration of either ABC294640 or ABC294735 to mice led to a delay in tumor growth in both xenograft models without overt toxicity to the animals. Tumor growth delay was potentiated by co-administration of sorafenib. These studies show that combination of an SK inhibitor with sorafenib causes synergistic inhibition of cell growth in vitro, and potentiates antitumor activity in vivo. Thus, a foundation is established for clinical trials evaluating the efficacy of combining these signaling inhibitors.
The sphingolipids ceramide, sphingosine, and sphingosine 1-phosphate (S1P) regulate cell signaling, proliferation, apoptosis, and autophagy. Sphingosine kinase-1 and -2 (SK1 and SK2) phosphorylate sphingosine to form S1P, shifting the balanced activity of these lipids toward cell proliferation. We have previously reported that pharmacological inhibition of SK activity delays tumor growth in vivo. The present studies demonstrate that the SK2-selective inhibitor 3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide (ABC294640) induces nonapoptotic cell death that is preceded by microtubule-associated protein light chain 3 cleavage, morphological changes in lysosomes, formation of autophagosomes, and increases in acidic vesicles in A-498 kidney carcinoma cells. ABC294640 caused similar autophagic responses in PC-3 prostate and MDA-MB-231 breast adenocarcinoma cells. Simultaneous exposure of A-498 cells to ABC294640 and 3-methyladenine, an inhibitor of autophagy, switched the mechanism of toxicity to apoptosis, but decreased the potency of the SK2 inhibitor, indicating that autophagy is a major mechanism for tumor cell killing by this compound. Induction of the unfolded protein response by the proteasome inhibitor N-(benzyloxycarbonyl)leucinylleucinylleucinal Z-Leu-Leu-Leu-al (MG-132) or the heat shock protein 90 inhibitor geldanamycin synergistically increased the cytotoxicity of ABC294640 in vitro. In severe combined immunodeficient mice bearing A-498 xenografts, daily administration of ABC294640 delayed tumor growth and elevated autophagy markers, but did not increase terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling-positive cells in the tumors. These data suggest that ABC294640 promotes tumor cell autophagy, which ultimately results in nonapoptotic cell death and a delay of tumor growth in vivo. Consequently, ABC294640 may effectively complement anticancer drugs that induce tumor cell apoptosis.
Sphingolipid-metabolizing enzymes control the dynamic balance of the cellular levels of important bioactive lipids, including the apoptotic compound ceramide and the proliferative compound sphingosine 1-phosphate (S1P). Many growth factors and inflammatory cytokines promote the cleavage of sphingomyelin and ceramide leading to rapid elevation of S1P levels through the action of sphingosine kinases (SK1 and SK2). SK1 and SK2 are overexpressed in a variety of human cancers, making these enzymes potential molecular targets for cancer therapy. We have identified an aryladamantane compound, termed ABC294640 [3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide], that selectively inhibits SK2 activity in vitro, acting as a competitive inhibitor with respect to sphingosine with a K(i) of 9.8 muM, and attenuates S1P formation in intact cells. In tissue culture, ABC294640 suppresses the proliferation of a broad panel of tumor cell lines, and inhibits tumor cell migration concomitant with loss of microfilaments. In vivo, ABC294640 has excellent oral bioavailability, and demonstrates a plasma clearance half-time of 4.5 h in mice. Acute and chronic toxicology studies indicate that ABC294640 induces a transient minor decrease in the hematocrit of rats and mice; however, this normalizes by 28 days of treatment. No other changes in hematology parameters, or gross or microscopic tissue pathology, result from treatment with ABC294640. Oral administration of ABC294640 to mice bearing mammary adenocarcinoma xenografts results in dose-dependent antitumor activity associated with depletion of S1P levels in the tumors and progressive tumor cell apoptosis. Therefore, this newly developed SK2 inhibitor provides an orally available drug candidate for the treatment of cancer and other diseases.
Intrinsic and acquired drug resistance remains a fundamental obstacle to successful applications of anticancer therapies for lung cancer. Combining conventional therapies with immunotherapeutic approaches is a promising strategy to circumvent lung cancer drug resistance. Genetically modified oncolytic viruses (OVs) kill tumor cells via completely unique mechanisms compared to small molecule chemotherapeutics typically used in lung cancer treatment and can also be used to deliver specific toxic, therapeutic or immunomodulatory genes to tumor cells. Recent pre-clinical and clinical studies with oncolytic vaccine approaches have revealed promising combination strategies that enhance oncolysis of tumor cells and circumvent tumor resistance mechanisms. As clinical trials with oncolytic vaccines progress, and as the knowledge acquired from these studies builds a foundation demonstrating OVs safety and efficacy, novel combination approaches could soon have a major impact on the clinical management of patients diagnosed with lung cancer.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.