Gα₁₂ Drives Invasion of Oral Squamous Cell Carcinoma through Up-Regulation of Proinflammatory Cytokines

<div><p>Oral squamous cell carcinoma (<i>OSCC</i>) ranks among the top ten most prevalent cancers worldwide. Like most head and neck squamous cell carcinomas (HNSCCs), OSCC is highly inflammatory and aggressive. However, the signaling pathways triggering the activation of its inflammatory processes remain elusive. G protein-coupled receptor signaling regulates the inflammatory response and invasiveness of cancers, but it remains unclear whether Gα₁₂ is a critical player in the inflammatory cytokine pathway during the tumorigenesis of OSCC. This study was undertaken to determine the role of Gα₁₂ signaling in the regulation of proinflammatory cytokines in their mediation of OSCC invasion. We found that both the transcription and protein levels of Gα₁₂ are up-regulated in OSCC tumors. The elevated Gα₁₂ expressions in OSCC patients also correlated with extra-capsular spread, an indicator of tumor invasiveness in HNSCCs. This clinical finding was supported by the studies of overexpression and RNAi knockdown of Gα₁₂ in OSCC cells, which demonstrated that Gα₁₂ promoted tumor cell migration and invasion. To understand how Gα₁₂ modulates OSCC invasiveness, we analyzed key biological processes in microarray data upon depletion of Gα₁₂ and found that cytokine- and other immune-related pathways were severely impaired. Importantly, the mRNA levels of IL-6 and IL-8 proinflammatory cytokines in clinical samples were found to be significantly correlated with the increased Gα₁₂ levels, suggesting a potential role of Gα₁₂ in modulating the IL-6 and IL-8 expressions. Supporting this hypothesis, overexpression or RNAi knockdown of Gα₁₂ in OSCC cell lines both showed that Gα₁₂ positively regulated the mRNA and protein levels of IL-6 and IL-8. Finally, we demonstrated that the Gα₁₂ promotion of tumor cell invasiveness was suppressed by the neutralization of IL-6 and IL-8 in OSCC cells. Together, these findings suggest that Gα₁₂ drives OSCC invasion through the up-regulation of IL-6 and IL-8 cytokines.</p></div

Temporal Transcription Program of Recombinant Autographa californica Multiple Nucleopolyhedrosis Virus

Baculoviruses, a family of large, rod-shaped viruses that mainly infect lepidopteran insects, have been widely used to transduce various cells for exogenous gene expression. Nonetheless, how a virus controls its transcription program in cells is poorly understood. With a custom-made baculovirus DNA microarray, we investigated the recombinant Autographa californica multiple nucleopolyhedrosis virus (AcMNPV) gene expression program in lepidopteran Sf21 cells over the time course of infection. Our analysis of transcription kinetics in the cells uncovered sequential viral gene expression patterns possibly regulated by different mechanisms during different phases of infection. To gain further insight into the regulatory network, we investigated the transcription program of a mutant virus deficient in an early transactivator (pe38) and uncovered several pe38-dependent and pe38-independent genes. This study of baculovirus dynamic transcription programs in different virus genetic backgrounds provides new molecular insights into how gene expression in viruses is regulated

Gα₁₂ promotes OSCC cell migration and invasion.

<p>(A) The transwell migration assay of Gα₁₂ overexpressed (Gα₁₂), or Gα₁₂ depleted (siGα₁₂) HSC-3 cells stained with crystal violet. The lower panel shows the quantitative results by three independent experiments. (B) The transwell invasion assay of Gα₁₂ overexpressed (Gα₁₂), or Gα₁₂ depleted (siGα₁₂) HSC-3 cells. The lower panel shows the quantitative results by three independent experiments. (C) Depletion of Gα₁₂ in two other OSCC cell lines (OC-3 and CGHNC9) also decreased cell migration and invasion. The knockdown efficiency and overexpression level of Gα₁₂ in four different OSCC cell lines used in this study are demonstrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066133#pone.0066133.s002" target="_blank">Figure S1</a>. The bottom panel shows the quantitative results. All the quantitative values were calculated at least in five distinct fields of each chamber. The data are expressed as a relative percentage to the controls. The statistic results were analyzed by <i>t-test</i>, *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001. Error bars represent the standard deviation (SD) of the mean from three independent experiments.</p

Transcriptome analysis reveals changes of immune-related pathways in OSCC and in Gα₁₂-depleted OSCC cell lines.

<p>(A) Comparative transcriptome analysis of OSCC tumors reveals that cytokine and other immune-related functional groups are listed in the top ten GO terms. A total of 1,616 differently expressed genes selected by 1.5 fold change cut-off (positive false discovery rate <i>q</i><10⁻⁸) in 55 OSCC tumors compared to 21 normal control tissues were analyzed by GO and pathway analysis tools. Functional groups of the inflammation-related pathways are highlight in red. (B) The immune-related signaling pathways are significantly impaired in the Gα₁₂-depleted OC-3 and HSC-3 cell lines. An arbitrary 2.0 fold-change cut-off is used to filter the differentially expressed genes compared between Gα₁₂-depleted and non-targeted siRNA control cells for the GO enrichment analysis. A total of 58 genes for HSC-3 cells and 218 genes for OC-3 cells were subjected to the analysis. The cytokine and interferon-mediated pathways (highlighted in red) were found in the GO terms for both cell lines. Detailed information of the GO terms is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066133#pone.0066133.s001" target="_blank">Table S1</a>.</p