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

The up-regulation of Gα₁₂ in OSCC patients correlates with Extra-capsular spread.

<p>(A) The Gα₁₂ expression is significantly up-regulated in 55 OSCC tumors compared to 21 normal control tissues (fold change >1.5, <i>P</i><10⁻¹⁰). The microarray data was analyzed by two-way clustering. Each column represents an individual clinical sample. Normalized gene expression values were color coded in percentage relative to the mean: blue for values less than the mean and red for values greater than the mean. (B) Quantitative RT-PCR (qPCR) analysis of Gα₁₂ in 25 OSCC tumors compared to 11 normal mucosa tissues. The results were normalized to GAPDH expression levels and then analyzed by <i>t-test</i>, **<i>P</i><0.01. Box plots display the median, 25th and 75th percentiles. Whiskers represent 5–95 percentiles and dots the outliers. (C) The box plot shows the relative gene expression values (RMA, log2) of Gα₁₂ for extra-capsular spread (ECS) positive (+) and negative (−) patients. Statistical results were analyzed by <i>t-test</i>, **<i>P</i><0.01. (D) Western blot analysis of Gα₁₂ levels in 6 paired samples of OSCC and adjacent normal/pre-cancerous tissues. The Gα₁₂ protein levels were found to be markedly up-regulated in OSCC tumor tissues compared to the GAPDH loading control. (E) Representative immunohistochemical images for Gα₁₂ staining patterns in the paraffin-embedded section of OSCC biopsies. Gα₁₂ immunoreactivity was detected primarily in the membrane and cytoplasm of OSCC (lower panel). In contrast, the adjacent normal and pre-cancerous oral tissues of individual patients showed very low immunoreactivity (upper panel). Original magnification, ×200.</p

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

Gα₁₂-dependent regulation of IL-6 and IL-8 in OSCC.

<p>(A) Dot plots of the linear regression analysis showing a positive correlation of gene expressions between Gα₁₂ and IL-6/IL-8 in OSCC tumors and normal mucosa tissues. The relative expression scales are shown by RMA value in the microarray data. (B) IL-6 and IL-8 mRNA levels are positively regulated by Gα₁₂ in OSCC cells. Gα₁₂ levels in four different OSCC cell lines (HSC-3, SCC25, OC3, and CGHNC9) were altered by the transient overexpression or RNAi knockdown of Gα₁₂. The qPCR results were normalized against GAPDH. The lower panel shows the electrophoresis image of the RT-PCR products. (C) The secreted proteins of IL-6 and IL-8 are up-regulated by Gα₁₂ in OSCC cells. ELISA assay was used to measure IL-6 and IL-8 in the conditioned media of the Gα₁₂-overexpressing or -depleted HSC-3, SCC25, OC-3, and CGHNC9 cells. (D) The LPA-induced IL-6 and IL-8 production is regulated by Gα₁₂. IL-6 and IL-8 protein levels in conditioned media of OSCC cells were measured by ELISA assay. The baseline IL-6 and IL-8 levels in conditioned media from four different OSCC cell lines are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066133#pone.0066133.s003" target="_blank">Figure S2</a>. All the quantitative results are expressed as a fold change relative to the controls. The statistical results were analyzed by <i>t-test</i>, *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001. “ns” means no significance. Error bars represent SD of the mean from three independent experiments.</p

MicroRNAs MiR-218, MiR-125b, and Let-7g Predict Prognosis in Patients with Oral Cavity Squamous Cell Carcinoma

<div><p>MicroRNAs (miRNAs) have a major impact on regulatory networks in human carcinogenesis. In this study, we sought to investigate the prognostic significance of miRNAs in patients with oral cavity squamous cell carcinoma (OSCC). In a discovery phase, RNA was extracted from 58 OSCC tumor samples and paired normal tissues. MiRNAs expression was evaluated with TaqMan Array Card and TaqMan MicroRNA assays. The prognostic significance of the miRNA signature identified in the discovery phase was validated by qRT-PCR in a replication set consisting of 141 formalin-fixed, paraffin-embedded (FFPE) samples. We identified a miRNA regulatory network centered on the three hub genes (<i>SP1</i>, <i>MYC</i>, and <i>TP53</i>) that predicted distinct clinical endpoints. Three miRNAs (miR-218, miR-125b, and let-7g) and their downstream response genes had a concordant prognostic significance on disease-free survival and disease-specific survival rates. In addition, patients with a reduced expression of miR-218, miR-125b, and let-7g have a higher risk of poor outcomes in presence of specific risk factors (p-stage III-IV, pT3-4, or pN+). Our findings indicate that specific miRNAs have prognostic significance in OSCC patients and may improve prognostic stratification over traditional risk factors.</p></div

Kaplan-Meier survival plots of OSCC patients according to traditional risk factors and miRNAs expression levels.

<p>(<b>A</b>) In the subgroup of patients with pT3-4 disease, subjects with high and low miR-125b expression had significantly different local control rates (92% vs. 50%); (<b>B</b>) the subgroup with low miR-218 expression showed a higher rate of distant metastases in patients with pN+ disease (86% vs. 57%); (<b>C</b>) a high let-7g expression was associated with a lower risk of tumor relapse in patients with advanced pathological stage (46% vs.71%); (<b>D</b>) an increased expression of let-7g predicted a better disease-specific survival in the subgroup of patients with pT3-4 disease (76% vs. 40%).</p

Multivariable analysis of 5-year control and survival rates in OSCC patients (<i>n</i> = 58).

<p>Abbreviations: HR, hazard ratio; CI, confidence interval; ns, not significant.</p><p>* Indicates risk factors significantly associated with outcomes in univariate analysis.</p

Prognostic miRNA modulators centered on the three hub genes <i>SP1</i>, <i>MYC</i>, and <i>TP53</i>.

<p>The downstream genes were grouped into outcome-specific clusters according to their prognostic significance (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102403#pone.0102403.s005" target="_blank">Tables S4</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102403#pone.0102403.s007" target="_blank">S6</a>). The associations between the upstream miRNAs and the downstream gene clusters were identified using sparse partial least square regression. Both the upstream miRNAs and the downstream responsive gene clusters showed a concordant prognostic impact on disease-free survival and disease-specific survival. The solid lines indicate the experimentally-confirmed physical interactions. The dotted lines show the results of the functional analysis performed using the DAVID package. <i>ABCA1</i>, <i>DDIT3</i>, <i>NDUFS8</i>, and <i>NDUFB9</i> regulate cell growth and proliferation. <i>TNFSF10</i> and <i>TNFRSF12A</i> are involved in the cell's apoptotic machinery. The remaining genes encode for molecules regulating cell adhesion or glycosaminoglycan metabolism. The miRNA modulators identified in this predicted poor outcomes in OSCC. Hopefully, our findings may lead to the development of novel prognostic models integrating molecular signatures and traditional risk factors for improving the prognostic stratification and the treatment modalities of OSCC patients.</p

DSG3 silencing increased the interaction of plakoglobin with the transcriptional factor TCF.

<p>(<b>A, B</b>) Knockdown of DSG3 increased plakoglobin binding to the TCF4 transcriptional factor, as determined by immunoprecipitation (IP) and immunoblot (IB) analysis as determined in OECM1 (A) and SAS (B) cells. Cellular extracts of the vector or the shDSG3 stable transfected cells were immunoprecipitated with anti-TCF4, rabbit IgG (IgG) (as a negative control) and subsequently immunoblotted with plakoglobin or β-catenin antibody. (<b>C</b>) Immunofluorescence and confocal microscopy were used to examine the interactions among plakpglobin and TCF4. The vector or shDGS3 stable transfected cells were co-stained with either plakoglobin and TCF4 antibodies. After washing, the slides were incubated with rhodamine- or FITC- conjugated second antibody. DAPI staining was performed for nuclear staining. The scale bar indicates the size as 40 µm.</p