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

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

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

Insights into fungal diversity of a shallow-water hydrothermal vent field at Kueishan Island, Taiwan by culture-based and metabarcoding analyses.

This paper reports the diversity of fungi associated with substrates collected at a shallow hydrothermal vent field at Kueishan Island, Taiwan, using both culture-based and metabarcoding methods. Culture of fungi from yellow sediment (with visible sulfur granules), black sediment (no visible sulfur granules), the vent crab Xenograpsus testudinatus, seawater and, animal egg samples resulted in a total of 94 isolates. Species identification based on the internal transcribed spacer regions of the rDNA revealed that the yellow sediment samples had the highest species richness with 25 species, followed by the black sediment (23) and the crab (13). The Ascomycota was dominant over the Basidiomycota; the dominant orders were Agaricales, Capnodiales, Eurotiales, Hypocreales, Pleosporales, Polyporales and Xylariales. Hortaea werneckii was the only common fungus isolated from the crab, seawater, yellow and black sediment samples. The metabarcoding analysis amplifying a small fragment of the rDNA (from 18S to 5.8S) recovered 7-27 species from the black sediment and 12-27 species from the yellow sediment samples and all species belonged to the Ascomycota and the Basidiomycota. In the yellow sediments, the dominant order was Pleosporales and this order was also dominant in the black sediment together with Sporidiobolales. Based on the results from both methods, 54 and 49 species were found in the black and yellow sediments, respectively. Overall, a higher proportion of Ascomycota (~70%) over Basidiomycota was recovered in the yellow sediment and the two phyla were equally abundant in the black sediment. The top five dominant fungal orders in descending order based on species richness were Pleosporales>Eurotiales>Polyporales>Hypocreales>Capnodiales in the black sediment samples, and Polyporales>Pleosporales>Eurotiales>Capnodiales>Hypocreales in the yellow sediment samples. This study is the first to observe a high diversity of fungi associated with various substrates at a marine shallow water hydrothermal vent ecosystem. While some fungi found in this study were terrestrial species and their airborne spores might have been deposited into the marine sediment, several pathogenic fungi of animals, including Acremonium spp., Aspergillus spp., Fusarium spp., Malassezia spp., Hortaea werneckii, Parengyodontium album, and Westerdykella dispersa, were recovered suggesting that these fungi may be able to cause diseases of marine animals

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field