The Effects Of CAMP/Epac1 Signal Pathway And Its Related Mechanisms On Novel Oncolytic Virus M1

Oncolytic virotherapy is a novel and emerging treatment modality that uses replication-competent viruses to selective destroy cancer cells. These natural-occuring or genetic engineered viruses selectively target cancer cells, without affecting the viabilities of normal cells. Oncolytic virotherapy obtained dramatic breakthroughs last year. Talimogene laherparepvec(T-Vec), an herpes simplex virus inserted with granulocyte macrophage colony-stimulatory factor(GM-CSF) through genetic engineering, is the first oncolytic virus used in a phase 3 clinical trial that demonstrated therapeutic benefit against melanoma and approved by FDA. Numerous studies have found that a growing number of viruses including poxvirus, adenovirus,vesicular stomatitis virus and reovirus can be adapted to cancer therapies for their restricted replication in tumor cells before or after engineering.Alphavirus M1 was isolated from a pool of culicine mosquitoes collected in Hainan island of China in 1964. Alphavirus M1 belongs to Togaviridae Getah-like virus, single-strand positive RNA virus, the genome is 11, 696 nucleotides(nt) in length. Previously, our group has identified that alphavirus M1 selectively targets Zinc-finger antiviral protein(ZAP) deficient cancer cells and induces prolonged and severed Endoplasmic Reticulum(ER) to mediate cell apoptosis. However, the sensitivity to oncolysis varies among different cancer cell lines. The varied sensitivities of M1 in different cancer cell lines drive us to explore the mechanisms and strategies, which can overcome the barriers.In this study, we sought to investigate the anticancer effectiveness of M1/cAMP combination treatment in vitro, in vivo and ex vivo. Mechanistically, we uncovered that cAMP activators blunt the antiviral response in tumor and promote viral replication. cAMP signaling pathway activators, as tumor-specific viral sensitizers,have the potential to remarkably increase the spectrum of malignancies amenable to oncolytic virotherapy and reactivate oncolytic virus M1 after intratumoral clearance.Methods and results:1. M1 infection inhibits the cell viability of large amounts of cancer cells.Methods: Human cancer cell lines, immortalized epithelial cell lines and primary normal cells; MTT assay for detection of cell viability; Phase-contrast microscopy for morphologic observation of M1 infected cells; TCID50 assay for virus titer determination.Results: The sensitivity of M1 varies in different cancer cells, with more viral replication in hypersensitive cancer cells and less viral replication in refractory cancer cells.2. M1 infection induces antiviral factor in refractory cancer cells, while cAMP signal pathway activation abrogates it.Methods: q RT-PCR for detection of the induced antiviral response factors. Western blot to determine the viral protein E1 expression.Results: M1 infection induces antiviral factors expression in refractory cancer cells.cAMP signal pathway activators abrogates it. Moreover, cAMP signal pathway activators abrogate the antiviral effects of recombinant Interferon-α.3. Activation of cAMP signal pathway cooperates with M1 to selectively kill cancer cells.Methods: MTT assays for the detection of the cell viabilities before or after db-cAMP treatment during M1 infection; TCID50 for the detection of the viral titer;Werstern blot for the detection of viral structural protein E1 and non-structural protein NS3.Results: M1 infection alone does not affect the cell viability of cancer cells HCT-116 and Capan-1 and normal cell L-02. Db-cAMP treatment enhances the oncolytic effects of M1 in many cancer cells, but it does not affect the cell viability of normal cells. Db-cAMP treatment increases the viral titer and viral proteins in cancer cells but not in normal cells.4. Enhanced oncolysis is mediated by prolonged and severe ER stress.Methods: Transmission electron microscope for observation of the organelle;Western blot for detection of the ER stress markers and ER stress mediated apoptosis pathways; Caspase activity kit for determination of the activity of Caspase-3/9.Results: We observed that M1/db-cAMP treatment induces swelling of ER.M1/db-cAMP treatment induces the ER stress markers Bip, IRE1α, PERK and phosphorylated e IF2α expression. M1/db-cAMP treatment activates ER stress mediated apoptosis pathway, JNK pathway, Chop pathway but not Caspase-12 pathway.5. Epac1 is necessary for cAMP enhanced viral oncolysis.Methods: Transfection of si RNA to knockdown the expression of PKA and Epac1;Western blot for detection of the expression of PKA or Epac1 and viral protein E1expression; MTT assay for the determination of the cell viability; TCID50 for determination of the viral titer.Results: We observed that knockdown of PKA does not affect the cell viabilities after M1/db-cAMP combination treatment. However, we observed that knockdown of Epac1 abrogates the enhanced oncolysis and increased viral protein expression by db-cAMP. Moreover, Epac1 specific inhibitor ESI-09 also can abrogates the enhanced oncolysis, increased viral titer and viral protein expression by db-cAMP.6. 8-CPT-cAMP inhibits tumor growth in combination with M1 and promotes virus replication in tumor.Methods: Subcutaneously inoculation of Hep3 B, HCT-116 and Capan-1 cancer cells in nude mice for the observation of the antitumor effects of 8-CPT-cAMP/M1 combination treatment; Tumor samples were dissected for the observation of the size and further immunohistochemistry(IHC) staining; q RT-PCR for the determination of the viral genome distribution in vivo; IHC for the observation of proliferation marker Ki-67 and apoptosis marker cleaved-Caspase-3;Results: We observed that 8-CPT-cAMP/M1 combination treatment significantly inhibited the tumor growth in vivo. 8-CPT-cAMP treatment specifically upregulates viral genome RNA in tumor. IHC staining indicates that proliferation marker Ki67 are significantly downregulated and apoptosis marker cleaved-Caspase-3 are significantly upregulated.7. 8-CPT-cAMP enhances oncolytic efficiency of M1 in primary human tumor specimens.Methods: TECIA for the test of the antitumor effects of M1/cAMP in Ex vivo model.Results: M1/cAMP combination treatment showed dramatic antitumor activities in three of four detected ex vivo model.Conclusions:1. M1 possesses antitumor effects in diverse range of cancer cells, while the sensitivity and viral replication levels vary in different cell lines.2. M1 virus infection induces antiviral factor expression in a time dependent manner,while db-cAMP and Forsklin abrogates the induced antiviral factor expression.3. In refractory cancer cells, cAMP pathway activators enhance the oncolytic effects of M1. The enhanced oncolysis is due to the increased viral replication in these cancer cells, indicating that cAMP pathway activators can be adapted to reactivation agents during M1 treatment.4. Enhanced oncolysis is due irresolvable ER stress. ER stress mediated apoptosis pathway JNK and CHOP pathways contribute to cell death.5. The enhanced oncolysis is not mediated by the classical cAMP dependent protein kinase A. The enhanced oncolysis is due to Epac1.6. M1/cAMP combination treatment inhibits tumor growth in vivo, with less Ki-67 expression and more cleaved-Caspase-3 expression. Moreover, cAMP specifically increases the viral genome RNA in tumor.7. M1/cAMP combination treatment inhibits tumor growth in ex vivo models,indicating that this combination treatment shows potential to enhance the oncolytic effects of M1 and increase the spectrum of antitumor therapies.