Extracellular Vesicles as Biomarkers in Head and Neck Squamous Cell Carcinoma: From Diagnosis to Disease-Free Survival.

Head and neck squamous cell carcinomas (HNSCCs) arising from different anatomical sites present with different incidences and characteristics, which requires a personalized treatment strategy. Despite the extensive research that has conducted on this malignancy, HNSCC still has a poor overall survival rate. Many attempts have been made to improve the outcomes, but one of the bottlenecks is thought to be the lack of an effective biomarker with high sensitivity and specificity. Extracellular vesicles (EVs) are secreted by various cells and participate in a great number of intercellular communications. Based on liquid biopsy, EV detection in several biofluids, such as blood, saliva, and urine, has been applied to identify the existence and progression of a variety of cancers. In HNSCC, tumor-derived EVs exhibit many functionalities by transporting diverse cargoes, which highlights their importance in tumor screening, the determination of multidisciplinary therapy, prediction of prognosis, and evaluation of therapeutic effects. This review illustrates the classification and formation of EV subtypes, the cargoes conveyed by these vesicles, and their respective functions in HNSCC cancer biology, and discloses their potential as biomarkers during the whole process of tumor diagnosis, treatment, and follow-up.

Convergent dysbiosis of upper aerodigestive microbiota between patients with esophageal and oral cavity squamous cell carcinoma.

The bidirectional association between primary esophageal squamous cell carcinoma (ESCC) and oral cavity squamous cell carcinoma (OSCC) suggests common risk factors and oncogenic molecular processes but it is unclear whether these two cancers display similar patterns of dysbiosis in their upper aerodigestive microbiota (UADM). We conducted a case-control study to characterize the microbial communities in esophageal lavage samples from 49 ESCC patients and oral rinse samples from 91 OSCC patients using 16S rRNA V3-V4 amplicon sequencing. Compared with their respective non-SCC controls from the same anatomical sites, 32 and 45 discriminative bacterial genera were detected in ESCC and OSCC patients, respectively. Interestingly, 20 of them were commonly enriched or depleted in both types of cancer, suggesting a convergent niche adaptation of upper aerodigestive SCC-associated bacteria that may play important roles in the pathogenesis of malignancies. Notably, Fusobacterium, Selenomonas, Peptoanaerobacter and Peptostreptococcus were enriched in both ESCC and OSCC, whereas Streptococcus and Granulicatelia were commonly depleted. We further identified Fusobacterium nucleatum as the most abundant species enriched in the upper aerodigestive SCC microenvironment, and the higher relative abundances of Selenomonas danae and Treponema maroon were positively correlated with smoking. In addition, predicted functional analysis revealed several depleted (eg, lipoic acid and pyruvate metabolism) and enriched (eg, RNA polymerase and nucleotide excision repair) pathways common to both cancers. Our findings reveal a convergent dysbiosis in the UADM between patients with ESCC and OSCC, suggesting a shared niche adaptation of host-microbiota interactions in the pathogenesis of upper aerodigestive tract malignancies.

Tumor-secreted exosomal miR-141 activates tumor-stroma interactions and controls premetastatic niche formation in ovarian cancer metastasis.

BACKGROUND: Metastatic colonization is one of the critical steps in tumor metastasis. A pre-metastatic niche is required for metastatic colonization and is determined by tumor-stroma interactions, yet the mechanistic underpinnings remain incompletely understood. METHODS: PCR-based miRNome profiling, qPCR, immunofluorescent analyses evaluated the expression of exosomal miR-141 and cell-to-cell communication. LC-MS/MS proteomic profiling and Dual-Luciferase analyses identified YAP1 as the direct target of miR-141. Human cytokine profiling, ChIP, luciferase reporter assays, and subcellular fractionation analyses confirmed YAP1 in modulating GROα production. A series of in vitro tumorigenic assays, an ex vivo model and Yap1 stromal conditional knockout (cKO) mouse model demonstrated the roles of miR-141/YAP1/GROα/CXCR1/2 signaling cascade. RNAi, CRISPR/Cas9 and CRISPRi systems were used for gene silencing. Blood sera, OvCa tumor tissue samples, and tissue array were included for clinical correlations. RESULTS: Hsa-miR-141-3p (miR-141), an exosomal miRNA, is highly secreted by ovarian cancer cells and reprograms stromal fibroblasts into proinflammatory cancer-associated fibroblasts (CAFs), facilitating metastatic colonization. A mechanistic study showed that miR-141 targeted YAP1, a critical effector of the Hippo pathway, reducing the nuclear YAP1/TAZ ratio and enhancing GROα production from stromal fibroblasts. Stromal-specific knockout (cKO) of Yap1 in murine models shaped the GROα-enriched microenvironment, facilitating in vivo tumor colonization, but this effect was reversed after Cxcr1/2 depletion in OvCa cells. The YAP1/GROα correlation was demonstrated in clinical samples, highlighting the clinical relevance of this research and providing a potential therapeutic intervention for impeding premetastatic niche formation and metastatic progression of ovarian cancers. CONCLUSIONS: This study uncovers miR-141 as an OvCa-derived exosomal microRNA mediating the tumor-stroma interactions and the formation of tumor-promoting stromal niche through activating YAP1/GROα/CXCRs signaling cascade, providing new insight into therapy for OvCa patients with peritoneal metastases.

Characterization of oral microbiota in HPV and non-HPV head and neck squamous cell carcinoma and its association with patient outcomes.

OBJECTIVE: To investigate the interplay among the oral microbiota, HPV infection, traditional risk factors and patient outcomes in head and neck squamous cell carcinoma (HNSCC). MATERIALS AND METHODS: A multi-center study of HNSCC patients with paired tumor and control tissues. We characterized the oral microbiota and HPV infection of tissues in 166 Chinese adults by sequencing the bacterial 16S rRNA V3-V4 and HPV L1 regions, respectively, and examined the associations among the oral microbiota, HPV and clinical features. RESULTS: A total of 15.7% of the surveyed HNSCC patients were positive for HPV DNA, with infection rates varying from 66.7% in oropharyngeal SCC to 10.4% in oral cavity SCC (OSCC). No HPV infection was detected in the surveyed hypopharyngeal SCC. HPV16 was largely the predominant type. HPV infection in non-OSCC, especially oropharyngeal SCC, was associated with advanced N stage and superior survival outcomes. Oral microbiota dysbiosis was observed in HNSCC tumors, with differentially abundant taxa mainly associated with HNSCC subtype, T stage, survival/relapse, HPV infection, and smoking. Notably, the enrichment of Fusobacterium in tumor tissues of OSCC patients was associated with no smoking, early T stage, early N stage, and better 3-year disease-specific survival. CONCLUSION: Our findings underscore the involvement of oral microbiota dysbiosis in OSCC pathogenesis, Fusobacterium is involved with improved OSCC patient outcomes, especially in patients lacking traditional risk factors. Understanding the complex interactions among the oral microbiota, HPV infection and other risk factors for HNSCC will provide important insights into the pathogenesis of HNSCC.

The Role of Arginine Metabolism in Oral Tongue Squamous Cell Carcinoma.

Early diagnosis and treatment do not prevent the high morbidity and poor prognosis of oral tongue squamous cell carcinoma (TSCC). Earlier studies have shown that ARG1 signaling is deregulated in TSCC. Here, we investigated the complexity of ARG1 metabolism in this cancer subsite to appreciate the therapeutic potential of this potential biological vulnerability. Various functional studies show that ARG1 overexpression in oral cancer cells inhibits cell proliferation and invasion compared with controls. Further, RNA-sequencing revealed numerous differentially expressed genes (DEGs) and associated networks were dysregulated by ARG1 overexpression, including hypoxia-inducible factor (HIFα) signaling, the natural killer cell signaling pathway and interferon signaling. Our work provides a foundation for understanding the mechanism of action of disrupted arginine metabolism in oral tongue squamous cell carcinoma. This may impact the community for developing further therapeutic approaches.

Proteomic Analysis of Circulating Extracellular Vesicles Identifies Potential Biomarkers for Lymph Node Metastasis in Oral Tongue Squamous Cell Carcinoma.

Lymph node metastasis is the most reliable indicator of a poor prognosis for patients with oral tongue cancers. Currently, there are no biomarkers to predict whether a cancer will spread in the future if it has not already spread at the time of diagnosis. The aim of this study was to quantitatively profile the proteomes of extracellular vesicles (EVs) isolated from blood samples taken from patients with oral tongue squamous cell carcinoma with and without lymph node involvement and non-cancer controls. EVs were enriched using size exclusion chromatography (SEC) from pooled plasma samples of patients with non-nodal and nodal oral tongue squamous cell carcinoma (OTSCC) and non-cancer controls. Protein cargo was quantitatively profiled using isobaric labelling (iTRAQ) and two-dimensional high-performance liquid chromatography followed by tandem mass spectrometry. We identified 208 EV associated proteins and, after filtering, generated a short list of 136 proteins. Over 85% of the EV-associated proteins were associated with the GO cellular compartment term "extracellular exosome". Comparisons between non-cancer controls and oral tongue squamous cell carcinoma with and without lymph node involvement revealed 43 unique candidate EV-associated proteins with deregulated expression patterns. The shortlisted EV associated proteins described here may be useful discriminatory biomarkers for differentiating OTSCC with and without nodal disease or non-cancer controls.

Profiling of extracellular vesicles in oral cancer, from transcriptomics to proteomics.

Oral cancers occurring in different subsites can have distinct etiologies' and are a significant problem worldwide. In general, the incidence of oral cancers has declined over the last decade due to improvements in modifiable risk factors (tobacco and alcohol consumption). However, recent data suggest that the incidence of squamous cell carcinomas in the oral tongue and oropharynx are increasing. Human papilloma virus (HPV) is an important risk factor for oropharyngeal cancer and is associated with better treatment responses when compared with HPV-unrelated oropharyngeal cancer. Regardless of the subsite, there are no clinically available biomarkers for the early detection of these cancers and many are detected at an advanced stage and are associated with poor 5-year survival rates. Tumor tissue and serial needle biopsies are used to diagnose and prognosticate oral cancers but have important limitations. Besides being invasive and physically painful, these types of biopsies offer a limited view of a complex tumor due to inter- and intra-tumoral heterogeneity and a dynamic tumor microenvironment. Liquid biopsies offer a promising and alternative way to measure disease in real-time. Extracellular vesicles (EVs) are small particles that are secreted by all cells types and can be readily isolated from a wide range of biofluids. EVs are structurally stable and can horizontally transfer bioactive molecules to distant sites throughout the body in concentrated forms that exceed what can be delivered in a soluble format. As EVs represent their cell of origin, biofluid derived EVs are heterogeneous and are comprised of a complex repertoire of host- and cancer-derived particles. This review article has focused on studies that have used transcriptomics and proteomics to explore the function and clinical significance of EVs in oral cavity and oropharyngeal cancers.

Targeting of lipid metabolism with a metabolic inhibitor cocktail eradicates peritoneal metastases in ovarian cancer cells.

Ovarian cancer is an intra-abdominal tumor in which the presence of ascites facilitates metastatic dissemination, and associated with poor prognosis. However, the significance of metabolic alterations in ovarian cancer cells in the ascites microenvironment remains unclear. Here we show ovarian cancer cells exhibited increased aggressiveness in ascites microenvironment via reprogramming of lipid metabolism. High lipid metabolic activities are found in ovarian cancer cells when cultured in the ascites microenvironment, indicating a metabolic shift from aerobic glycolysis to ß-oxidation and lipogenesis. The reduced AMP-activated protein kinase (AMPK) activity due to the feedback effect of high energy production led to the activation of its downstream signaling, which in turn, enhanced the cancer growth. The combined treatment of low toxic AMPK activators, the transforming growth factor beta-activated kinase 1 (TAK1) and fatty acid synthase (FASN) inhibitors synergistically impair oncogenic augmentation of ovarian cancer. Collectively, targeting lipid metabolism signaling axis impede ovarian cancer peritoneal metastases.

A deliberative framework to identify the need for real-life evidence building of new cancer drugs after interim funding decision.

BACKGROUND: With the rising cost of new oncology treatments, it is no longer sustainable to base initial drug funding decisions primarily on prospective clinical trials as their performance in real-life populations are often difficult to determine. In British Columbia, an approach in evidence building is to retrospectively analyse patient outcomes using observational research on an ad hoc basis. METHODS: The deliberative framework was constructed in three stages: framework design, framework validation and treatment programme characterization, and key informant interview. Framework design was informed through a literature review and analyses of provincial and national decision-making processes. Treatment programmes funded between 2010 and 2013 were used for framework validation. A selection concordance rate of 80% amongst three reviewers was considered to be a validation of the framework. Key informant interviews were conducted to determine the utility of this deliberative framework. RESULTS: A multi-domain deliberative framework with 15 assessment parameters was developed. A selection concordance rate of 84.2% was achieved for content validation of the framework. Nine treatment programmes from five different tumour groups were selected for retrospective outcomes analysis. Five contributory factors to funding uncertainties were identified. Key informants agreed that the framework is a comprehensive tool that targets the key areas involved in the funding decision-making process. CONCLUSIONS: The oncology-based deliberative framework can be routinely used to assess treatment programmes from the major tumour sites for retrospective outcomes analysis. Key informants indicate this is a value-added tool and will provide insight to the current prospective funding model.

Tumour growth-suppressive effect of arsenic trioxide in squamous cell lung carcinoma.

Lung squamous cell carcinoma (SCC) is the second most common subtype of non-small cell lung carcinoma. The anticancer effects of arsenic trioxide (ATO) in lung adenocarcinoma and small-cell lung cancer have previously been reported; however its effects in SCC remain unclear. An MTT assay and western blot analysis were performed to determine cell viability and protein expression, respectively, in the SK-MES-1 and SW900 SCC cell lines following treatment with ATO. Phosphatidylserine externalization, mitochondrial membrane depolarization and cell cycle distribution were studied using flow cytometry and the in vivo effects of ATO on tumour growth were investigated with a xenograft model. The results demonstrated that SK-MES-1 and SW900 SCC cells were sensitive to clinically relevant concentrations of ATO. ATO induced apoptosis, mitochondrial membrane depolarization and G2/M arrest. In addition, treatment with ATO resulted in the downregulation of X-linked inhibitor of apoptosis, B-cell lymphoma-2 (Bcl-2), E2F transcription factor 1 (E2F1), thymidylate synthase and ribonucleotide reductase M1 in addition to the upregulation of Bcl-2 antagonist/killer protein, cleaved poly ADP-ribose polymerase and cleaved caspase 3 in a cell-line specific manner. In the SW900 xenograft model, tumour growth was inhibited by ATO with the formation of apoptotic bodies and downregulation of Bcl-2 and E2F1. In conclusion, ATO suppresses the growth of SCC in vitro and in vivo.

Growth suppressive effect of pegylated arginase in malignant pleural mesothelioma xenografts.

BACKGROUND: Malignant pleural mesothelioma (MPM) is a difficult-to-treat global disease. Pegylated arginase (BCT-100) has recently shown anti-tumor effects in hepatocellular carcinoma, acute myeloid leukemia and melanoma. This study aims to investigate the effects of PEG-BCT-100 in MPM. METHODS: A panel of 5 mesothelioma cell lines (H28, 211H, H226, H2052 and H2452) was used to study the in vitro effects of BCT-100 by crystal violet staining. The in vivo effects of BCT-100 were studied using 211H and H226 nude mice xenografts. Protein expression (argininosuccinate synthetase, ornithine transcarbamylase, cleaved PARP, cleaved caspase 3, cyclins (A2, D3, E1 and H), CDK4 and Ki67) and arginine concentration were evaluated by Western blot and ELISA respectively. Cellular localization of BCT-100 was detected by immunohistochemistry and immunoflorescence. TUNEL assay was used to identify cellular apoptotic events. RESULTS: Argininosuccinate synthetase was expressed in H28, H226, and H2452 cells as well as 211H and H266 xenografts. Ornithine transcarbamylase was undetectable in all cell lines and xenograft models. BCT-100 reduced in vitro cell viability (IC50 values at 13-24 mU/ml, 72 h) across different cell lines and suppressed tumor growth in both 211H and H226 xenograft models. BCT-100 (60 mg/kg) significantly suppressed tumor growth (p < 0.01) with prolonged median survival (p < 0.01) in both xenograft models. Combining BCT-100 with pemetrexed or cisplatin conferred no additional benefits over single agents. Serum and intratumoral arginine levels were effectively decreased by BCT-100, associated with cytosolic accumulation of BCT-100 within tumor cells. Apoptosis (PARP cleavage in 211H xenografts; Bcl-2 downregulation, and cleavage of PARP and caspase 3 in H226 xenografts; positive TUNEL staining in both) and G1 arrest (downregulation of cyclin A2, D3, E1 and CDK4 in 211H xenografts; suppression of cyclin A2, E1, H and CDK4 in H226 xenografts) were evident with BCT-100 treatment. Furthermore, proliferative factor Ki67 was downregulated in BCT-100 treatments arms. CONCLUSIONS: BCT-100 suppressed tumor growth with prolonged median survival partially mediated by intratumoral arginine depletion resulting in apoptosis and G1 arrest in mesothelioma xenograft models. The findings provide scientific evidence to support further clinical development of BCT-100 in treatment of MPM.

MicroRNA-141 enhances anoikis resistance in metastatic progression of ovarian cancer through targeting KLF12/Sp1/survivin axis.

BACKGROUND: Cancer metastasis is determined by the formation of the metastatic niche and the ability of cancer cells to adapt to microenvironmental stresses. Anoikis resistance is a fundamental feature of metastatic cancer cell survival during metastatic cancer progression. However, the mechanisms underlying anoikis resistance in ovarian cancer are still unclear. METHODS: Expressions of miRNA-141 and its downstream targets were evaluated by qPCR, Western blotting, Immunohistochemical (IHC) and in situ hybridization (ISH) assays. The luciferase assays were used to prove KLF12 as the downstream target of miR-141. The cDNA microarray and apoptotic protein arrays were used to identify the targets of miR-141 and KLF12. The competition of KLF12 and Sp1 on survivin promoter was examined by ChIP assay. IHC analysis on ovarian cancer tissue array was used to evaluate the expressions of KLF12 and miR-141 and to show the clinical relevance. The functional studies were performed by in vitro and in vivo tumorigenic assays. RESULTS: Enforced expression of miR-141 promotes, while knockdown of miR-141 expression inhibits, cell proliferation, anchorage-independent capacity, anoikis resistance, tumor growth and peritoneal metastases of ovarian cancer cells. Bioinformatics and functional analysis identified that Kruppel-related zinc finger protein AP-2rep (KLF12) is directly targeted by miR-141. Consistent with this finding, knockdown of KLF12 phenocopied the effects of miR-141 overexpression in ovarian cancer cells. In contrast, restoration of KLF12 in miR-141-expressing cells significantly attenuated anoikis resistance in ovarian cancer cells via interfering with Sp1-mediated survivin transcription, which inhibits the intrinsic apoptotic pathway and is crucial for ovarian cancer cell survival, anoikis resistance and peritoneal metastases. Immunohistochemical (IHC) and in situ hybridization (ISH) assays confirmed that miRNA-141 expression is inversely correlated with KLF12 expression and significantly associated with advanced ovarian cancers accompanied with distal metastases, underscoring the clinical relevance of our findings. CONCLUSIONS: Our data identify a novel signaling axis of miR-141/KLF12/Sp1/survivin in enhancing anoikis resistance and likely serves as a potential therapeutic target for metastatic ovarian cancer.

Combination effects of arsenic trioxide and fibroblast growth factor receptor inhibitor in squamous cell lung carcinoma.

OBJECTIVES: Lung cancer remains the top cancer killer worldwide, with squamous cell carcinoma (SCC) as the second commonest histologic subtype. Arsenic trioxide (ATO) was previously shown to suppress growth of lung cancer. Fibroblast growth factor receptor (FGFR) amplification was recently demonstrated in lung SCC, with specific FGFR inhibitor (e.g. PD173074) developed as a potential targeted therapy. Therefore the combination effects of ATO and PD173074 in SCC was studied. MATERIALS AND METHODS: The combination of ATO/PD173074 was studied in a proof-of-principle model using a lung SCC cell line with FGFR1 overexpression: SK-MES-1. The effects of ATO and/or PD173074 on cell viability and protein expression were studied by MTT assay and Western blot respectively. Cell cycle analysis, phosphatidylserine externalization and mitochondrial membrane depolarization were monitored by flow cytometry. FGFR1 knockdown was performed with siRNAs. Proteasome inhibitor (MG-132) was used to study the degradation mechanism. In vivo effect of ATO and/or PD173074 was investigated using a nude mice xenograft model. RESULTS: Combined ATO/PD173074 reduced cell viability along with increased sub-G1 population, phosphatidylserine externalization and mitochondrial membrane depolarization more significantly than single treatments. Downregulation of FGFR1, p-Akt, Akt, p-Src, Src, p-c-Raf, c-Raf, Erk and survivin as well as upregulation of p-Erk and cleaved PARP were observed upon ATO and/or PD treatment. MG-132 partially reversed the degradation of Akt, Src, c-Raf and Erk induced by ATO/PD, suggestive of ubiquitin-independent proteasome-dependent degradation. However, the mechanism of FGFR1 downregulation remained unknown. Downregulation of FGFR1, Akt, Src, c-Raf and Erk as well as cleaved PARP elevation induced by ATO and/or PD were confirmed in vivo. CONCLUSION: Massive protein degradation (FGFR1, Akt, Src, c-Raf and Erk) was induced by ATO and/or PD173074 treatment mainly mediated by activation of proteasomal degradation in SCC cell line SK-MES-1 in vitro and in vivo.

E2F1 downregulation by arsenic trioxide in lung adenocarcinoma.

Lung cancer is one of the most common cancers worldwide. Arsenic trioxide (ATO) has been approved by the US Food and Drug Administration for the treatment of acute promyelocytic leukemia. Nonetheless preliminary data have suggested potential activity of ATO in solid tumors including lung cancer. This study aimed to examine the underlying mechanisms of ATO in the treatment of lung adenocarcinoma. Using a panel of 7 lung adenocarcinoma cell lines, the effects of ATO treatment on cell viability, expression of E2F1 and its downstream targets, phosphatidylserine externalization, mitochondrial membrane depolarization and alteration of apoptotic/anti-apoptotic factors were studied. Tumor growth inhibition in vivo was investigated using a nude mouse xenograft model. ATO decreased cell viability with clinically achievable concentrations (8 µM) in all cell lines investigated. This was accompanied by reduced expression of E2F1, cyclin A2, skp2, c-myc, thymidine kinase and ribonucleotide reductase M1, while p-c-Jun was upregulated. Cell viability was significantly decreased with E2F1 knockdown. Treatment with ATO resulted in phosphatidylserine externalization in H23 cells and mitochondrial membrane depolarization in all cell lines, associated with truncation of Bid, downregulation of Bcl-2, upregulation of Bax and Bak, caspase-9 and -3 activation and PARP cleavage. Using the H358 xenograft model, the tumor growth was suppressed in the ATO treatment group during 8 days of treatment, associated with downregulation of E2F1 and upregulation of truncated Bid and cleaved caspase-3. In conclusion, ATO has potent in vitro and in vivo activity in lung adenocarcinoma, partially mediated through E2F1 downregulation and apoptosis.