Adenoviruses harboring the herpes simplex virus thymidine kinase (HSVtk) gene under the regulation of a trans-splicing ribozyme targeting human telomerase reverse transcriptase (hTERT-TR) show marked and specific antitumor activity. In addition to inducing tumor cell death by direct cytotoxicity, it is becoming clear that HSVtk also induces antitumor immunity. Programmed death ligand 1 (PD-L1) expressed on tumor cell surfaces mediates tumor-induced immunoresistance by inhibiting PD1-expressing tumor-infiltrating T cells. Here, we explored whether a soluble form of PD1 (sPD1-Ig), which blocks PD-L1, could synergize with TERT-TR-regulated HSVtk to enhance the adenoviral therapeutic efficacy by boosting antitumor immunity. Tumor antigen released by HSVtk-transduced tumors successfully primed tumor antigen–specific CD8 T cells via dendritic cells (DC). Regression of murine tumors was markedly enhanced when sPD1-Ig was incorporated into the adenovirus as compared with a single-module adenovirus expressing only HSVtk. This effect was abolished by CD8 T-cell depletion. Consistent with this, following adoptive transfer of tumor antigen–specific CD8 T cells into tumor-bearing Rag1^−/− mice, dual-module adenovirus significantly enhanced CD8 T cell–mediated tumor rejection. In addition, secondary tumor challenge at a distal site was completely suppressed in mice treated with a dual-module adenovirus. These results suggest that a dual-targeting strategy to elicit both tumor antigen priming and tumor-induced immunoresistance enhances CD8 T cell–mediated antitumor immunity.

C-MYC is a promising target for cancer therapy but its use is restricted by unwanted, devastating side effects. We explored whether intravesical instillation of the c-MYC inhibitor KSI-3716 could suppress tumor growth in murine orthotopic bladder xenografts. The small molecule KSI-3716, which blocks c-MYC/MAX binding to target gene promoters, was used as an intravesical chemotherapy agent. KSI-3716 action was assessed by electrophoretic mobility shift assay, chromatin immunoprecipitation, transcription reporter assay and quantitative reverse transcriptase-polymerase chain reaction. Inhibition of cell proliferation and its mechanism was monitored by cell cytotoxicity assay, EdU incorporation assay and flow cytometry. The in vivo efficacy of KSI-3716 was examined by noninvasive luminescence imaging and histological analysis after intravesical instillation of KSI-3716 in murine orthotopic bladder xenografts. KSI-3716 blocked c-MYC/MAX from forming a complex with target gene promoters. c-MYC mediated transcriptional activity was inhibited by KSI-3716 at concentrations as low as 1 μM. The expression of c-MYC target genes, such as cyclin D2, CDK4 and hTERT, was markedly decreased. KSI-3716 exerted cytotoxic effects on bladder cancer cells by inducing cell cycle arrest and apoptosis. Intravesical instillation of KSI-3716 at a dose of 5 mg/kg significantly suppressed tumor growth with minimal systemic toxicity. The c-MYC inhibitor KSI-3716 could be developed as an effective intravesical chemotherapy agent for bladder cancer.

Antitumor activity of the c-Myc inhibitor KSI-3716 in gemcitabine-resistant bladder cancer Antitumor activity of the c-Myc inhibitor KSI-3716 in gemcitabine-resistant bladder cancer

CD46 is generally overexpressed in many human cancers, representing a prime target for CD46-binding adenoviruses (Ads). This could help to overcome low anti-tumoral activity by coxsackie-adenoviral receptor (CAR)-targeting cancer gene therapy viruses. However, because of scarce side-by-side information about CAR and CD46 expression levels in cancer cells, mixed observations of cancer therapeutic efficacy have been observed. This study evaluated Ad-mediated therapeutic efficacy using either CAR-targeting Ad5 or CD46-targeting Ad5/35 fiber chimera in bladder cancer cell lines. Compared with normal urothelia, bladder cancer tissue generally overexpressed both CAR and CD46. While CAR expression was not correlated with disease progression, CD46 expression was inversely correlated with tumor grade, stage, and risk grade. In bladder cancer cell lines, expression levels of CD46 and CAR were highly correlated with Ad5/35- and Ad5-mediated gene transduction and cytotoxicity, respectively. In a human EJ bladder cancer xenograft mouse model, with either overexpressed or suppressed CD46 expression levels, Ad5/35-tk followed by ganciclovir (GCV) treatment significantly affected tumor growth, whereas Ad5-tk/GCV had only minimal effects. Overall, our findings suggest that bladder cancer cells overexpress both CAR and CD46, and that adenoviral cancer gene therapy targeting CD46 represents a more suitable therapy option than a CAR-targeting therapy, especially in patients with low risk bladder cancers.

Studies have shown that insertion of oleic acid into lipid bilayers can modulate the membrane properties of liposomes so as to improve their function as drug carriers. Considering that 2-hydroxyoleic acid (2OHOA), a potential antitumor agent currently undergoing clinical trials, is a derivative of oleic acid, we explored the possibility of developing 2OHOA-inserted liposomes as a multifunctional carrier of antitumor drugs in the present study. The insertion of 2OHOA into lipid bilayers was confirmed by surface charge determination and differential scanning calorimetry. 2OHOA insertion greatly decreased the order of dimyristoylphosphatidylcholine packing, produced a nanosized (<100 nm) dispersion, and improved the colloidal stability of liposomes during storage. Moreover, 2OHOA-inserted liposome forms exhibited greater growth inhibitory activity against cancer cells compared with free 2OHOA, and the growth-inhibitory activity of liposomal 2OHOA was selective for tumor cells. 2OHOA insertion greatly increased the liposome-incorporated concentration of hydrophobic model drugs, including mitoxantrone, paclitaxel, and all-trans retinoic acid (ATRA). The in vitro anticancer activity of ATRA-incorporated/2OHOA-inserted liposomes was significantly higher than that of ATRA-incorporated conventional liposomes. In a B16-F10 melanoma syngeneic mouse model, the tumor growth rate was significantly delayed in mice treated with ATRA-incorporated/2OHOA-inserted liposomes compared with that in the control group. Immunohistochemical analyses revealed that the enhanced antitumor activity of ATRA-incorporated/2OHOA-inserted liposomes was due, at least in part, to increased induction of apoptosis. Collectively, our findings indicate that 2OHOA-inserted liposomes exhibit multiple advantages as antitumor drug carriers, including the ability to simultaneously deliver two anticancer drugs – 2OHOA and incorporated drug – to the tumor tissue.