Fig 2: CCND1, CCND2, CDK2 and CCNE2 mediates the miR-150-downregulation-induced NPC cell growth. (A) Individual silencing of CCND1, CCND2, CDK2 and CCNE2 rescued the proliferation rate enhanced by anti-miR-150. (B) Individual silencing of CCND1, CCND2, CDK2 and CCNE2 rescued the colony-formation ability enhanced by anti-miR-150. (C) Individual silencing of CCND1, CCND2, CDK2 and CCNE2 rescued the anchorage-independent growth ability enhanced by anti-miR-150.

Fig 3: Attenuation of CDK expression and cyclin D-CDK4/6 activity by androgen in HPr-1AR.(A) HPr-1AR cells were treated with 10 nM DHT or vehicle control for 24 hours, and CDK2, CDK4 and CDK6 mRNAs and CDKN1A and CDKN1B mRNAs were quantified using QPCR. CDK4 and CDK6 mRNAs were androgen-repressed 2–3 fold. CDKN1A mRNA was DHT-induced 2-fold whereas CDKN1B mRNA was unresponsive to DHT. Data represent the mean ± SEM, n = 3. (B) HPr-1AR cells were treated with several DHT doses (10−11, 10−10, 10−9, 10−8 M) or vehicle control for 24 hours, and significant decreases in CDK4 and CDK6 mRNAs occurred at DHT doses ranging from 0.1–10 nM, n = 3. These DHT doses also increased CDKN1A mRNA. (C) AR antagonist 2-hydroxyflutamide (OHF, 10 μM) suppressed the down-regulation of CDK mRNAs and the up-regulation of CDKN1A mRNA by 1 nM DHT treatment at 24 hours, n = 3. (D) HPr-1AR cells were treated with 10 nM DHT or vehicle control for 24–48 hours (h). Immunoblot analysis of cell lysates reveals that CDK4 and CDK6 protein expression decreased and CDKN1A expression increased with DHT treatment compared to vehicle. CDK2 phosphorylation at threonine 160 (T160) decreased nearly 50% with androgen treatment at 24 and 48 hours, albeit not significantly, and total CDK2 protein expression remained unchanged. Relative to total RB expression, the levels of various phosphorylated forms of RB (S780 and S807/811) decreased over time with androgen treatment. (E) DHT treatment significantly decreased CDK4 and CDK6 protein levels 55% or more at 24 hours and 65% or more at 48 hours. The phosphorylated forms of RB declined 55% or more at 24 hours and 65% or more at 48 hours. Relative CDK/GAPDH and p-RB/total RB protein ratios are shown using the mean ± SEM, n = 4. * P < 0.05.

Fig 4: Effects of siCD44, cisplatin or their combination on cell cycle distribution of H460 cells. H460 cells were seeded into 6-well plate at 1.5×105 cells/well and transfected with sictr and siCD44 respectively for 24 h, followed by treatment of cisplatin at concentration of 10 µM for another 24 h. (A) Transfected cells were stained with PI for cell cycle analysis by flow cytometry. Cell distribution was analyzed by MFLT32. (B) Corresponding statistical analysis of cell distribution. (C) The protein expressions of CD44, c-Myc, CDK2, CDK4, CDK6 and cyclin D1 in H460 were analyzed by western blotting, with GAPDH as loading control. Data were presented as mean ± SD of 3 independent experiments. *P<0.05 vs. sictr; #P<0.05 vs. sictr + cisplatin. sictr, control siRNA; siCD44, CD44 siRNA; PI, propidium iodide.

Fig 5: Model of interaction between an oncolytic virus and the host type I IFN response and cyclin expression. Host pattern recognition receptors (PRR) will recognize intracellular viral particles and initiate a type I IFN response. Upon viral recognition, IFN signals through the type I IFNα/β receptor (IFNAR) leading to the activation of Janus kinase 1 and/or tyrosine kinase 2 (JAK1/TYK2) and recruitment of signal transducer and activator of transcription 1 (STAT1) for phosphorylation and dimerization. The phosphorylated STAT1 dimer then binds IRF9 to form the interferon-stimulated gene factor 3 complex (ISGF3) complex, which binds IFN-stimulated response elements located in ISGs, resulting in the activation of antiviral genes. Some of the most well-studied antiviral genes include OAS1 and MxA. OAS1 is dsRNA-dependent synthetase that activates the endoribonuclease RNase L to degrade ssRNA while MxA proteins bind target viral proteins to inhibit viral replication. The expression of these ISGs induces an antiviral state. Viruses have developed mechanisms to antagonize the type I IFN response. The ability of E1A protein to decrease IRF9 levels, thereby inhibiting the type I IFN pathway, is shown. Viral E1A protein promotes cyclin E expression, which in turn, binds to and promotes the activation of CDK2. Activated CDK2 promotes viral replication. Palbociclib increases IRF9 expression and promotes cyclin E/CDK2 activation. Thus, the ability of the oncolytic virus to replicate and cause cytotoxicity will be dependent on the level of activation of type I IFN and cyclin E/CDK2.