Identification of a serum-based microRNA signature that detects recurrent oral squamous cell carcinoma before it is clinically evident.

BACKGROUND: Survival rates for oral squamous cell carcinoma (OSCC) have remained poor for decades, a fact largely attributable to late-stage diagnoses and high recurrence rates. We report analysis of serum miRNA expression in samples from patients with high-risk oral lesions (HRL, including OSCC/carcinoma in situ lesions) and healthy non-cancer controls, with the aim of non-invasively detecting primary or recurrent disease before it is clinically evident. METHODS: Discovery, test, and validation sets were defined from a total of 468 serum samples (305 HRL and 163 control samples). Samples were analysed using multiple qRT-PCR platforms. RESULTS: A two-miRNA classifier comprised of miR-125b-5p and miR-342-3p was defined following discovery and test analyses. Analysis in an independent validation cohort reported sensitivity and specificity of ~74% for this classifier. Significantly, when this classifier was applied to serial serum samples taken from patients both before treatment and during post-treatment surveillance, it identified recurrence an average of 15 months prior to clinical presentation. CONCLUSIONS: These results indicate this serum miRNA classifier is effective as a simple, non-invasive monitoring tool for earlier detection of recurrent disease when lesions are typically smaller and amenable to a wider array of treatment options to improve survival.

Extracellular vesicle secretion of miR-142-3p from lung adenocarcinoma cells induces tumor promoting changes in the stroma through cell-cell communication.

Extracellular vesicles (EVs) are mediators of communication between cancer cells and the surrounding tumor microenvironment. EV content is able to influence key tumorigenic changes including invasion, metastasis, and inducing pro-tumor changes in the stroma. MiR-142-3p is a known tumor suppressor in LAC and was recently shown to be enriched within LAC EVs, indicating its potential as a key signaling miRNA. Our research demonstrates the role EV associated miR-142-3p plays when transferred from LAC cells to both endothelial and fibroblast cells. We demonstrate that transfer of miR-142-3p in LAC EVs to endothelial cells promotes angiogenesis through inhibition of TGFßR1. Additionally, we show EV associated miR-142-3p promotes the cancer-associated fibroblast phenotype in lung fibroblast cells which we show is independent of TGFß signaling. These findings suggest that miR-142-3p within LAC EVs can be transferred from LAC cells to both endothelial and fibroblast cells to promote tumor associated changes.

Epigenetic mediated silencing of EYA4 contributes to tumorigenesis in oral dysplastic cells.

Five-year survival rates for oral squamous cell carcinoma (OSCC) have remained at a dismal 50% for the past several decades. Molecular analyses of premalignant tissues are a key means of identifying early foundational drivers of disease, which may be exploitable as biomarkers or therapeutic targets for improving disease outcomes. We previously identified EYA4 as frequently hypermethylated and silenced in premalignant disease based on an analysis of lesion-adjacent normal, dysplasia, and carcinoma in situ/squamous cell carcinoma tissues from the oral cavity. Herein, we further evaluate the role of this putative tumor suppressor gene in transformation of oral tissues and OSCC. By an initial assessment, EYA4 promoter hypermethylation was found in 24/32 (75%) of paired tumor samples in The Cancer Genome Atlas oral cancer data set, with significant correlation noted between methylation status and relative gene expression. To assess the impact of EYA4 in oral tumorigenesis, we overexpressed EYA4 in two oral dysplasia cell lines. Expression of EYA4 caused an increase in cell proliferation, DNA damage repair capabilities, and increased the level of apoptosis. Taken together, we find evidence that EYA4 is a novel tumor suppressor in oral cancer, which becomes methylated and silenced at the premalignant stage and appears to be epigenetically regulated. Further studies are warranted to investigate its role as a marker for progression in oral cancer. © 2016 Wiley Periodicals, Inc.

Molecular characterization of immortalized normal and dysplastic oral cell lines.

BACKGROUND: Cell lines have been developed for modeling cancer and cancer progression. The molecular background of these cell lines is often unknown to those using them to model disease behaviors. As molecular alterations are the ultimate drivers of cell phenotypes, having an understanding of the molecular make-up of these systems is critical for understanding the disease biology modeled. METHODS: Six immortalized normal, one immortalized dysplasia, one self-immortalized dysplasia, and two primary normal cell lines derived from oral tissues were analyzed for DNA copy number changes and changes in both mRNA and miRNA expression using SMRT-v.2 genome-wide tiling comparative genomic hybridization arrays, Agilent Whole Genome 4x44k expression arrays, and Exiqon V2.M-RT-PCR microRNA Human panels. RESULTS: DNA copy number alterations were detected in both normal and dysplastic immortalized cell lines-as well as in the single non-immortalized dysplastic cell line. These lines were found to have changes in expression of genes related to cell cycle control as well as alterations in miRNAs that are deregulated in clinical oral squamous cell carcinoma tissues. Immortal lines-whether normal or dysplastic-had increased disruption in expression relative to primary lines. All data are available as a public resource. CONCLUSIONS: Molecular profiling experiments have identified DNA, mRNA, and miRNA alterations for a panel of normal and dysplastic oral tissue cell lines. These data are a valuable resource to those modeling diseases of the oral mucosa, and give insight into the selection of model cell lines and the interpretation of data from those lines.

Extracellular vesicles promote activation of pro-inflammatory cancer-associated fibroblasts in oral cancer.

Introduction: Oral squamous cell carcinoma (OSCC) is the most common form of head and neck cancer and has a survival rate of ∼50% over 5 years. New treatment strategies are sorely needed to improve survival rates-and a better understanding of the mechanisms underlying tumorigenesis is needed to develop these strategies. The role of the tumor microenvironment (TME) has increasingly been identified as crucial in tumor progression and metastasis. One of the main constituents of the TME, cancer-associated fibroblasts (CAFs), plays a key role in influencing the biological behavior of tumors. Multiple mechanisms contribute to CAF activation, such as TGFß signaling, but the role of extracellular vesicles (EVs) in CAF activation in OSCC is poorly understood. Assessing the impact of oral cancer-derived EVs on CAF activation will help to better illuminate OSCC pathophysiology and may drive development of novel treatments options. Methods: EVs were isolated from OSCC cell lines (Cal 27, SCC-9, SCC-25) using differential centrifugation. Nanoparticle tracking analysis was used for EV characterization, and Western blot to confirm the presence of EV protein markers. Oral fibroblasts were co-cultured with enriched EVs, TGFß, or PBS over 72 h to assess activation. Flow cytometry was used to evaluate CAF markers. RNA collected from fibroblasts was extracted and the transcriptome was sequenced. Conditioned media from the co-cultures was evaluated with cytokine array profiling. Results: OSCC-derived EVs can activate oral fibroblasts into CAFs that are different from those activated by TGFß, suggesting different mechanisms of activation and different functional properties. Gene set enrichment analysis showed several upregulated inflammatory pathways in those CAFs exposed to OSCC-derived EVs. Marker genes for inflammatory CAF subtypes were also upregulated, but not in CAFs activated by TGFß. Finally, cytokine array analysis on secreted proteins revealed elevated levels of several pro-inflammatory cytokines from EV-activated CAFs, for instance IL-8 and CXCL5. Discussion: Our results reveal the ability of OSCC-derived EVs to activate fibroblasts into CAFs. These CAFs seem to have unique properties, differing from TGFß-activated CAFs. Gaining an understanding of the interplay between EVs and stromal cells such as CAFs could lead to further insights into OSCC tumorigenesis and potential novel therapeutics.

Sphingomyelin phosphodiesterase 3 methylation and silencing in oral squamous cell carcinoma results in increased migration and invasion and altered stress response.

Neutral sphingomyelinase 2 (nSMase2), the product of the sphingomyelin phosphodiesterase 3 (SMPD3) gene, catalyzes the hydrolysis of sphingomyelin to ceramide. Ceramide acts on various signaling pathways to influence cell proliferation, survival, and stress response. Altered levels of sphingolipids and ceramides have been reported in various cancer types, including oral squamous cell carcinoma (OSCC). OSCC patients exhibit a poor 5-year survival rate of 50%, a figure that has remained stagnant for decades. To overcome this requires a better understanding of the molecular events driving this disease. The molecular analysis of the oral cavity reported here has identified the SMPD3 promoter region as a site of frequent hypermethylation and downregulation in pre-malignant and malignant tissues as compared with healthy control tissues. While lentivirus-induced overexpression of SMPD3 in cell models of oral dysplasia and OSCC did not significantly alter proliferation, it did decrease migration and invasion and increased resistance to the epidermal growth factor receptor (EGFR) inhibitor erlotinib. These results suggest that SMPD3 downregulation is a common event in OSCC progression and may promote the spread of tumor cells.

The Role of Extracellular Vesicles in Cancer: Cargo, Function, and Therapeutic Implications.

Extracellular vesicles (EVs) are a heterogeneous collection of membrane-bound structures that play key roles in intercellular communication. EVs are potent regulators of tumorigenesis and function largely via the shuttling of cargo molecules (RNA, DNA, protein, etc.) among cancer cells and the cells of the tumor stroma. EV-based crosstalk can promote proliferation, shape the tumor microenvironment, enhance metastasis, and allow tumor cells to evade immune destruction. In many cases these functions have been linked to the presence of specific cargo molecules. Herein we will review various types of EV cargo molecule and their functional impacts in the context of oncology.

Selective secretion of microRNAs from lung cancer cells via extracellular vesicles promotes CAMK1D-mediated tube formation in endothelial cells.

Extracellular vesicles (EVs) are key signaling mediators between cancer cells and their supporting stroma, and regulate critical processes such as invasion, metastases, and angiogenesis. We have identified a subset of miRNAs (miR-142-3p, miR-143-3p, miR-145-5p, miR-150-5p, miR-223-3p, miR-451a, miR-486-5p, miR-605-5p) that are enriched in lung adenocarcinoma extracellular vesicles compared to the donor cells from which they were derived. Two well-known tumor suppressors, miR-143-3p and miR-145-5p, were also enriched in serum samples collected during surgery from blood vessels draining directly from lung adenocarcinoma tumor beds. Recently, both miRNAs were found to promote neoangiogenesis in endothelial cells in mouse models of lung adenocarcinoma through targeting of CAMK1D, an inhibitory kinase that can impair angiogenesis when over-expressed. We show that the transfer of miR-143-3p and miR-145-5p within extracellular vesicles from lung adenocarcinoma cells to endothelial cells reduces the levels of CAMK1D and increases tube formation by endothelial cells. This finding suggests that transfer of miRNAs within extracellular vesicles is a method of communication between cancer and endothelial cells which promotes angiogenesis while simultaneously removing tumor suppressive miRNAs within the tumor cells, thus driving tumorigenesis.

Detection of clinically relevant copy number alterations in oral cancer progression using multiplexed droplet digital PCR.

Copy number alterations (CNAs), a common genomic event during carcinogenesis, are known to affect a large fraction of the genome. Common recurrent gains or losses of specific chromosomal regions occur at frequencies that they may be considered distinctive features of tumoral cells. Here we introduce a novel multiplexed droplet digital PCR (ddPCR) assay capable of detecting recurrent CNAs that drive tumorigenesis of oral squamous cell carcinoma. Applied to DNA extracted from oral cell lines and clinical samples of various disease stages, we found good agreement between CNAs detected by our ddPCR assay with those previously reported using comparative genomic hybridization or single nucleotide polymorphism arrays. Furthermore, we demonstrate that the ability to target specific locations of the genome permits detection of clinically relevant oncogenic events such as small, submicroscopic homozygous deletions. Additional capabilities of the multiplexed ddPCR assay include the ability to infer ploidy level, quantify the change in copy number of target loci with high-level gains, and simultaneously assess the status and viral load for high-risk human papillomavirus types 16 and 18. This novel multiplexed ddPCR assay therefore may have clinical value in differentiating between benign oral lesions from those that are at risk of progressing to oral cancer.

Recurring DNA copy number gain at chromosome 9p13 plays a role in the activation of multiple candidate oncogenes in progressing oral premalignant lesions.

Genomic alteration at chromosome 9p has been previously reported as a frequent and critical event in oral premalignancy. While this alteration is typically reported as a loss driven by selection for CDKN2A deactivation (at 9p21.3), we detect a recurrent DNA copy number gain of ~2.49 Mbp at chromosome 9p13 in oral premalignant lesions (OPLs) that later progressed to invasive lesions. This recurrent alteration event has been validated using fluorescence in situ hybridization in an independent set of OPLs. Analysis of publicly available gene expression datasets aided in identifying three oncogene candidates that may have driven selection for DNA copy number increases in this region (VCP, DCTN3, and STOML2). We performed in vitro silencing and activation experiments for each of these genes in oral cancer cell lines and found that each gene is independently capable of upregulating proliferation and anchorage-independent growth. We next analyzed the activity of each of these genes in biopsies of varying histological grades that were obtained from a diseased oral tissue field in a single patient, finding further molecular evidence of parallel activation of VCP, DCTN3, and STOML2 during progression from normal healthy tissue to invasive oral carcinoma. Our results support the conclusion that DNA gain at 9p13 is important to the earliest stages of oral tumorigenesis and that this alteration event likely contributes to the activation of multiple oncogene candidates capable of governing oral cancer phenotypes.

Global analysis of DNA methylation changes during progression of oral cancer.

OBJECTIVES: Earlier studies involving a priori gene selection have identified promoter regions deregulated by DNA methylation changes in oral squamous cell cancers (OSCCs) and precancers. Interrogation of global DNA methylation patterns for such specimens has not been reported, though such analyses are needed to uncover novel molecular factors driving disease. MATERIALS AND METHODS: We evaluated global DNA methylation patterns for 30 biopsies obtained from 10 patients undergoing surgical removal of an OSCC or carcinoma in situ (CIS). From a disease field in each patient, we collected (i) dysplastic, (ii) CIS or OSCC, and (iii) adjacent normal biopsies. DNA isolated from each biopsy was profiled for methylation status using the Illumina HumanMethylation27K platform. RESULTS: Our data demonstrate that aberrant methylation of promoter CpG islands exists across oral precancer and OSCC genomes. Non-hierarchical clustering of all methylation data revealed distinct methylation patterns between the normal and the CIS/OSCC tissues (with results for dysplastic biopsies split between groups). Multiple genes exhibiting recurrent aberrant DNA methylation were found for both dysplastic and CIS/OSCC groups, and included enrichment for genes found in the WNT and MAPK signaling pathways. CONCLUSION: In identifying aberrant DNA methylation at the earliest stages of oral precancer and finding recurring epigenetic disruption of specific genes/pathways across our analyzed cohort, we see evidence that CpG methylation changes may play a role in oral cancer progression and that global DNA methylation analyses may have significant utility in wider studies that seek to derive biomarkers or potentially druggable targets to improve oral cancer outcomes.

Methylation-mediated molecular dysregulation in clinical oral malignancy.

Herein we provide a concise review of the state of methylation research as it pertains to clinical oral cancerous and precancerous tissues. We provide context for ongoing research efforts in this field and describe technologies that are presently being applied to analyze clinical specimens. We also discuss the various recurrent methylation changes that have been reported for oral malignancy (including those genes frequently silenced by promoter methylation and the small RNAs with activity modulated by methylation changes) and describe surrogate disease markers identified via epigenetic analysis of saliva and blood specimens from patients with oral cancer.