Proteomics Analysis Reveals Involvement of Krt17 in Areca Nut-Induced Oral Carcinogenesis.

The areca nut is a known carcinogen that causes oral cancer in individuals in Southeast Asia, but the molecular mechanism that leads to this malignancy is still unclear. To mimic the habit of areca nut chewing, our laboratory has established four oral cancer cell sublines (SAS, OECM1, K2, C9), which have been chronically exposed to areca nut extract (ANE). To elucidate the molecular basis of areca nut-induced oral carcinogenesis, the differential proteomes between oral cancer cells and the ANE-treated sublines were determined using isobaric mass tag (iTRAQ) labeling and multidimensional liquid chromatography-mass spectrometry (LC-MS/MS). Over 1000 proteins were identified in four sublines, and 196 proteins were found to be differentially expressed in at least two ANE-treated sublines. A bioinformatic analysis revealed that these proteins participate in several pathways, and one of the most prominent pathways was the regulation of epithelial to mesenchymal transition (EMT). In all, 24 proteins including Krt17 were confirmed to be differentially expressed in the ANE-treated sublines. To reveal additional information on the mechanism of ANE-induced carcinogenesis, Krt17 was further investigated. Krt17 knockdown significantly suppressed ANE-induced cell migration and invasion and modulated the EMT process. Furthermore, in a murine model of carcinogen-induced (arecoline cocktail, an active compound of ANE) oral cancer, Krt17 was significantly up-regulated in all hyperplastic tissues and in carcinoma tissues (p < 0.001). In conclusion, we have identified a proteome of oral cancer cells that is associated with chronic areca nut exposure. Krt17 was demonstrated to contribute to areca nut-induced oral malignancy. The results of this study contribute to risk assessment, disease prevention and other clinical applications associated with areca nut-induced oral cancer.

Areca nut contributes to oral malignancy through facilitating the conversion of cancer stem cells.

Oral cancer is one of the most frequent malignant diseases worldwide, and areca nut is a primary carcinogen causing this cancer in Southeast Asia. Previous studies to examine the effects of this carcinogen often used short-term and high-dose treatment of area nut extract as a research model, which do not recapitulate the conditions of patients with long-term and habitual use of this substance. To approach authentic mechanism of areca nut-induced oral carcinogenesis that occurs in human, we established four isogenic sublines of oral cells which were chronic exposed to areca nut extract. Without eliciting cytotoxicity or senescence, these four sublines cells exhibited significant increase in invasive ability, along with epithelial-mesenchymal transition. These cells also showed resistance to chemotherapeutic drug and irradiation, accompanying with the augmentation of ABCG2 protein efflux and increased ROS clearance. Moreover, these sublines possessed the characteristics of cancer stemness, as demonstrated by enriched CD24-/CD44+ and CD133+ sub-populations, enhanced spheroid cell formation, and induced expressions of pluripotent stemness regulators, including Gp96, Grp78, Slug, Sox9, Snail, and Foxc2. These stemness regulators were further shown up-regulations in oral cancer patients with areca nut-chewing habit, and were statistically correlated with CD44 expression, a stemness marker. In conclusion, our findings suggested that areca nut contributes to oral malignancy through facilitating the conversion of cancer stem cells. This study may further contribute to clinical applications in disease prevention, risk assessment or molecular therapeutics on areca nut- associated diseases.

Fascin is a circulating tumor marker for head and neck cancer as determined by a proteomic analysis of interstitial fluid from the tumor microenvironment.

BACKGROUND: Head and neck cancer (HNC) is a prevalent cancer worldwide; however, clinically useful tumor markers for HNC have not been identified. Here, we aimed to identify secretory proteins from the tumor microenvironment as candidate circulating tumor markers. METHODS: Samples derived from seven pairs of tumor interstitial fluid (TIF) and normal interstitial fluid (NIF) samples from patients with HNC were analyzed. The proteomes were determined by gel-based-mass-spectrometry proteomic methods. The most up-regulated protein, fascin was confirmed in the cancer tissues and cell culture supernatant by immunoblotting and immunohistochemistry assays. Serum fascin was determined in 40 HNC and 40 normal individuals by ELISA. RESULTS: After proteomics analysis, 189 peptides were identified, corresponding to 75 proteins. Of the 21 proteins which were identified more than twice, five up-regulated proteins identified most frequently including fascin. The most elevated fascin was over-expressed in cancer tissues and cell culture supernatant. Serum fascin was significantly up-regulated in the cancer patients (p<0.001) and correlated with pathological lymph node metastasis (p=0.022). To assess the diagnostic efficacy, serum levels of fascin and another potential biomarker SCCA were determined. Fascin showed a high predictable value with an area under the curve (AUC) of 0.808 (95% CI 0.723-0.901) in the receiver operator curve (ROC), compared to 0.501 (95% CI 0.378-0.634) for SCCA. CONCLUSIONS: We have identified 75 potential circulating tumor markers associated with HNC, including fascin. Serum fascin could discriminate cancer patients from healthy individuals; thus, it may serve as a circulating biomarker for HNC.

Reciprocal regulation of microRNA-99a and insulin-like growth factor I receptor signaling in oral squamous cell carcinoma cells.

BACKGROUND: MicroRNAs (miRNAs), small noncoding RNA molecules can function as oncogenes or tumor suppressors in tumorigenesis. Oral squamous cell carcinoma (OSCC) is one of the most prevalent cancers worldwide with a 5-year survival rate of approximately 50%. METHODS: The expression of microRNA-99a (miR-99a) in OSCC tissues and cell lines was investigated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. The functions of miR-99a in migration/invasion and lung colonization were determined by transwell and tail vein injection assays, respectively. Specific targets of miR-99a were determined by software prediction, correlation with target protein expression, and luciferase reporter assay. The signaling pathways involved in regulation of miR-99a were investigated using the kinase inhibitors. RESULTS: We observed reduced levels of miR-99a, identified as one of the most downregulated miRNA in OSCC and all tested OSCC cell lines compared to normal oral keratinocytes. Ectopic miR-99a expression in OSCC cells markedly reduced migration and invasion in vitro as well as lung colonization in vivo. When evaluating the specific targets of miR-99a, we found that ectopic miR-99a expression downregulates insulin-like growth factor 1 receptor (IGF1R) protein and that the expression of miR-99a correlates negatively with IGF1R protein in OSCC cells. Insertion of the 3'UTR of IGF1R mRNA into the 3'UTR of a reporter gene markedly reduced luciferase activity in OSCC cells expressing miR-99a, suggesting that miR-99a reduces luciferase activity by targeting the 3'UTR of IGF1R mRNA. When evaluating the mechanisms of miR-99a downregulation, we observed the upregulation of miR-99a expression in serum-starved conditions and its suppression in response to insulin-like growth factor (IGF1) stimulation. Inhibitors of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) kinase inhibited IGF1-induced suppression of miR-99a, suggesting the negative regulation of miR-99a expression by IGF1R signaling. CONCLUSION: Overall, results indicate that miR-99a functions as a tumor metastasis suppressor in OSCC cells and mutually regulates IGF1R expression in a reciprocal regulation.

OncomiR-196 promotes an invasive phenotype in oral cancer through the NME4-JNK-TIMP1-MMP signaling pathway.

BACKGROUND: MicroRNA-196 (miR-196), which is highly up-regulated in oral cancer cells, has been reported to be aberrantly expressed in several cancers; however, the significance of miR-196 in oral cancer has not yet been addressed. METHODS: Cellular functions in response to miR-196 modulation were examined, including cell growth, migration, invasion and radio/chemosensitivity. Algorithm-based studies were used to identify the regulatory target of miR-196. The miR-196 target gene and downstream molecular mechanisms were confirmed by RT-qPCR, western blot, luciferase reporter and confocal microscopy analyses. miR-196 expression was determined in paired cancer and adjacent normal tissues from oral cancer patients. RESULTS: Both miR-196a and miR-196b were highly over-expressed in the cancer tissue and correlated with lymph node metastasis (P = 0.001 and P = 0.006, respectively). Functionally, miR-196 actively promoted cell migration and invasion without affecting cell growth. Mechanistically, miR-196 performed it's their function by inhibiting NME4 expression and further activating p-JNK, suppressing TIMP1, and augmenting MMP1/9. CONCLUSION: miR-196 contributes to oral cancer by promoting cell migration and invasion. Clinically, miR-196a/b was significantly over-expressed in the cancer tissues and correlated with lymph node metastasis. Thus, our findings provide new knowledge of the underlying mechanism of cancer metastasis. miR-196 may serve as a promising marker for better oral cancer management.

Systemic Investigation Identifying Salivary miR-196b as a Promising Biomarker for Early Detection of Head-Neck Cancer and Oral Precancer Lesions.

BACKGROUND: Liquid biopsy is a rapidly growing field, for it may provide a minimally invasive way to acquire pathological data for personalized medicine. This study developed a systemic strategy to discover an effective salivary biomarker for early detection of patients with head-neck squamous carcinoma (HNSC) and oral precancer lesion (OPC). METHODS: A total of 10 miRNAs were examined in parallel with multiple independent cohorts. These included a training set of salivary samples from HNSC patients, the TCGA-HNSC and GSE31277 cohorts to differentiate miRNAs between tumor and normal tissues, and groups of salivary samples from healthy individuals, patients with HNSC and OPC. RESULTS: The combined results from the salivary training set and the TCGA-HNSC cohort showed that four miRNAs (miR-148b, miR-155, miR-196b, and miR-31) consistently increased in HNSC patients. Further integration with the GSE31277 cohort, two miRNAs (miR-31 and miR-196b) maintained at high significances. Further assessment showed that salivary miR-196b was a prominent diagnostic biomarker, as it remarkably discriminated between healthy individuals and patients with HNSC (p < 0.0001, AUC = 0.767, OR = 5.64) or OPC (p < 0.0001, AUC = 0.979, OR = 459). CONCLUSION: Salivary miR-196b could be an excellent biomarker for diagnosing OPC and early detection of HNSC. This molecule may be used for early screening high-risk groups of HNSC.

Upregulated Expression of MicroRNA-204-5p Leads to the Death of Dopaminergic Cells by Targeting DYRK1A-Mediated Apoptotic Signaling Cascade.

MicroRNAs (miRs) downregulate or upregulate the mRNA level by binding to the 3'-untranslated region (3'UTR) of target gene. Dysregulated miR levels can be used as biomarkers of Parkinson's disease (PD) and could participate in the etiology of PD. In the present study, 45 brain-enriched miRs were evaluated in serum samples from 50 normal subjects and 50 sporadic PD patients. The level of miR-204-5p was upregulated in serum samples from PD patients. An upregulated level of miR-204-5p was also observed in the serum and substantia nigra (SN) of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. Expression of miR-204-5p increased the level of α-synuclein (α-Syn), phosphorylated (phospho)-α-Syn, tau, or phospho-tau protein and resulted in the activation of endoplasmic reticulum (ER) stress in SH-SY5Y dopaminergic cells. Expression of miR-204-5p caused autophagy impairment and activation of c-Jun N-terminal kinase (JNK)-mediated apoptotic cascade in SH-SY5Y dopaminergic cells. Our study using the bioinformatic method and dual-luciferase reporter analysis suggests that miR-204-5p positively regulates mRNA expression of dual-specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) by directly interacting with 3'UTR of DYRK1A. The mRNA and protein levels of DYRK1A were increased in SH-SY5Y dopaminergic cells expressing miR-204-5p and SN of MPTP-induced PD mouse model. Knockdown of DYRK1A expression or treatment of the DYRK1A inhibitor harmine attenuated miR-204-5p-induced increase in protein expression of phospho-α-Syn or phospho-tau, ER stress, autophagy impairment, and activation of JNK-mediated apoptotic pathway in SH-SY5Y dopaminergic cells or primary cultured dopaminergic neurons. Our results suggest that upregulated expression of miR-204-5p leads to the death of dopaminergic cells by targeting DYRK1A-mediated ER stress and apoptotic signaling cascade.

Head and neck cancer in the betel quid chewing area: recent advances in molecular carcinogenesis.

Head and neck cancer (HNC) is one of the 10 most frequent cancers worldwide, with an estimated over 500,000 new cases being diagnosed annually. The overall 5-year survival rate in patients with HNC is one of the lowest among common malignant neoplasms and has not significantly changed during the last two decades. Oral cavity squamous cell carcinoma (OSCC) shares part of HNC and has been reported to be increasing in the betel quid chewing area in recent years. During 2006, OSCC has become the sixth most common type of cancer in Taiwan, and it is also the fourth most common type of cancer among men. It follows that this type of cancer wreaks a high social and personal cost. Environmental carcinogens such as betel quid chewing, tobacco smoking and alcohol drinking have been identified as major risk factors for head and neck cancer. There is growing interest in understanding the relationship between genetic susceptibility and the prevalent environmental carcinogens for HNC prevention. Within this review, we discuss the molecular and cellular aspects of HNC carcinogenesis in Taiwan, an endemic betel quid chewing area. Knowledge of molecular carcinogenesis of HNC may provide critical clues for diagnosis, prognosis, individualization of therapy and molecular therapeutics.

Piperlongumine inhibits cancer stem cell properties and regulates multiple malignant phenotypes in oral cancer.

Piperlongumine (PL), a natural product of Piper longum, inhibits multiple malignant phenotypes. Therefore, the present study examined whether PL suppresses cancer stemness in oral cancer. The cellular effects of PL were determined by examining alterations in tumor sphere formation, cell migration, invasion, proliferation ability, chemosensitivity and radiosensitivity. Reverse transcription-quantitative polymerase chain reaction analysis and western blotting were performed in order to determine molecular expression levels. The present study revealed that PL inhibited cancer stem cell-forming ability and suppressed the expression of the stemness-related transcription factors SRY-Box 2, POU class 5 homeobox 1, and Nanog homeobox. However, it increased the expression of the differentiation marker cytokeratin 18. PL also suppressed cell migration and invasion, resulting in the elimination of the epithelial-mesenchymal transition. Furthermore, PL increased chemo- and radiosensitivity and suppressed tumor growth in vitro and in vivo. The results of the present study suggested that PL inhibits malignant phenotypes via the suppression of cancer stemness in oral cancer. Thus, PL may serve as an effective therapeutic agent for oral cancer.

MiR-520b as a novel molecular target for suppressing stemness phenotype of head-neck cancer by inhibiting CD44.

Cancer stem cells preferentially acquire the specific characteristics of stress tolerance and high mobility, allowing them to progress to a therapy-refractive state. To identify a critical molecule to regulate cancer stemness is indispensable to erratically cure cancer. In this study, we identified miR-520b as a novel molecular target to suppress head-neck cancer (HNC) with stemness phenotype. MiR-520b inhibited cellular migration and invasion via the mechanism of epithelial-mesenchymal transition. It also sensitized cells to therapeutic drug and irradiation. Significantly, miR-520b suppressed spheroid cell formation, as well as reduced expressions of multiple stemness regulators (Nestin, Twist, Nanog, Oct4). The CD44 molecule was identified as a direct target of miR-520b, as shown by the reverse correlative expressions, the response to miR-520 modulation, the luciferase reporter assay, and the functional rescue analyses. These cellular results were confirmed by a tumor xenograft mice study. Administration of miR-520b dramatically restrained tumorigenesis and liver colonization. Conversely, miR-520b silencing led to an acceleration of tumor growth. Taken together, our study demonstrated that miR-520b inhibits the malignancy of HNC through regulation of cancer stemness conversion by targeting CD44. MiR-520b may serve as an emerging therapeutic target that may be further developed for the intervention of refractory HNC.

GDF15 contributes to radioresistance and cancer stemness of head and neck cancer by regulating cellular reactive oxygen species via a SMAD-associated signaling pathway.

Radiotherapy is an integral part for the treatment of head and neck cancer (HNC), while radioresistance is a major cause leads to treatment failure. GDF15, a member of the TGF-ß superfamily, is hypothesized to participate in various types of homeostasis. However, the potential role of this molecule in regulation of radiosensitivity remains unclear. In this study, we demonstrated that GDF15 contributed to radioresistance of HNC, as determined by both gain- and lost-of-functional experiments. These results were achieved by the induction of mitochondrial membrane potential and suppression of intracellular reactive oxygen species (ROS). We further showed that GDF15 facilitated the conversion of cancer stemness, as assessed by the promotion of CD44+ and ALDH1+ cell populations and spheroid cell formation. At molecular level, GDF15 conferred to these cellular functions was through phosphorylated SMAD1 proteins to elite downstream signaling molecules. These cellular results were further confirmed in a tumor xenograft mouse study. Taken together, our results demonstrated that GDF15 contributed to radioresistance and cancer stemness by regulating cellular ROS levels via a SMAD-associated signaling pathway. GDF15 may serve as a prediction marker of radioresistance and a therapeutic target for the development of radio-sensitizing agents for the treatment of refractory HNC.

miR-196, an Emerging Cancer Biomarker for Digestive Tract Cancers.

Over the past decade, the emergence of microRNA (miRNA) research has firmly established this molecular family as a key component in cells. MiRNAs, which function as negative gene regulators, participate in multiple biological processes and maintain homeostasis in cells. The dysregulation of miRNA may contribute to numerous human disorders, including cancer. Recently, miR-196 was found to be aberrantly expressed in a wide range of malignant diseases, which suggests that it plays important roles in carcinogenesis. Here, we summarize the current knowledge concerning miR-196 family in cancers. This review includes miR-196 gene structure and aberrant expression in various cancers, and current understanding of numerous functions and regulatory targets of miR-196 in specific cancers. Since miR-196 are consistently found over-expressed in digestive tract cancer tissues, we also reviewed the clinical significance and potential applications of miR-196 in these cancers. We highlight that miR-196 may serve as an emerging cancer biomarker for digestive tract cancers.

Transketolase Serves a Poor Prognosticator in Esophageal Cancer by Promoting Cell Invasion via Epithelial-Mesenchymal Transition.

Background: To characterize the potential function and clinical significance of Transketolase (TKT) in esophageal cancer. Methods: High invasive esophageal squamous cell carcinoma (ESCC) cell line CE48T/VGH was used. Cellular functions in response to TKT modulation were examined, including cell growth, migration and invasion. The underlying molecules involved in the TKT regulatory mechanism were determined by western blot and confocal microscopic analysis. Clinically, TKT expressions in 76 ESCC patients were assessed by immunohistochemical (IHC) method, and the association with treatment outcome was determined. Results: TKT silencing inhibited cell migration and invasion but had a minimal effect on cell growth. This TKT silencing also induced the reversion of epithelial-mesenchymal transition (EMT), as evidenced by the spindle to cuboidal morphological change, increased the expression of epithelial markers (γ-catenin), and decreased the levels of mesenchymal markers (fibronectin and N-cadherin). Mechanically, TKT was shown to modulate the EMT through the pERK-Slug/Snail-associated signaling pathway. Clinically, a high level of TKT in the cancer tissues of patients with esophageal squamous cell carcinoma was associated with poor survival (P = 0.042). In the multivariate analysis, a high TKT level was also shown to be an independent unfavorable prognostic factor (Odds ratio: 1.827, 95% confidence interval: 1.045-3.196, P = 0.035). Conclusions: TKT contributes to esophageal cancer by promoting cell invasion via meditating EMT process. Clinically, the over-expression of TKT in ESCC patients predicts poorer survival. TKT inhibition may be a useful strategy to intervene in cancer cell invasion and metastasis, which may lead to better prognosis for ESCC patients.

Combined determination of circulating miR-196a and miR-196b levels produces high sensitivity and specificity for early detection of oral cancer.

OBJECTIVES: The aim of this study was to determine whether the oncogenic microRNA family members miR-196a and miR-196b can be circulating biomarkers for the early detection of oral cancer. DESIGN AND METHODS: To determine the stability of circulating miRNA, the blood sample was aliquot and stored at different temperature conditions for analysis. To assess the diagnostic efficacy, we determined the levels of miR-196s in plasma samples, including 53 from healthy individuals, 16 from pre-cancer patients, and 90 from oral cancer patients. RESULTS: In general, circulating miRNA was very stable when storing plasma samples at -20°C or below. In clinical study, both circulating miR-196a and miR-196b were substantially up-regulated in patients with oral pre-cancer lesions (5.9- and 14.8-fold, respectively; P < 0.01), as well as in oral cancer patients (9.3- and 17.0-fold, respectively; P < 0.01). These results show prominent discrimination between normal and pre-cancer patients (AUC = 0.764 or 0.840, miR-196a or miR-196b, respectively), and between normal and cancer patients (AUC = 0.864 or 0.960, miR-196a or miR-196b, respectively). The combined determination of miR-196a and miR-196b levels produces excellent sensitivity and specificity in the diagnosis of patients with oral pre-cancer (AUC = 0.845) or oral cancer (AUC = 0.963), as well as in the prediction of potential malignancy (AUC = 0.950, sensitivity = 91%, specificity = 85%). CONCLUSION: Combined determination of circulating miR-196a and miR-196b levels may serve as panel plasma biomarkers for the early detection of oral cancer.

miRNA-491-5p and GIT1 serve as modulators and biomarkers for oral squamous cell carcinoma invasion and metastasis.

MicroRNAs offer tools to identify and treat invasive cancers. Using highly invasive isogenic oral squamous cell carcinoma (OSCC) cells, established using in vitro and in vivo selection protocols from poorly invasive parental cell populations, we used microarray expression analysis to identify a relative and specific decrease in miR-491-5p in invasive cells. Lower expression of miR-491-5p correlated with poor overall survival of patients with OSCCs. miR-491-5p overexpression in invasive OSCC cells suppressed their migratory behavior in vitro and lung metastatic behavior in vivo. We defined the G-protein-coupled receptor kinase-interacting protein 1 (GIT1)-as a direct target gene for miR-491-5p control. GIT1 overexpression was sufficient to rescue miR-491-5p-mediated inhibition of migration/invasion and lung metastasis. Conversely, GIT1 silencing phenocopied the ability of miR-491-5p to inhibit migration/invasion and metastasis of OSCC cells. Mechanistic investigations indicated that miR-491-5p overexpression or GIT1 attenuation reduced focal adhesions, with a concurrent decrease in steady-state levels of paxillin, phospho-paxillin, phospho-FAK, EGF/EGFR-mediated extracellular signal-regulated kinase (ERK1/2) activation, and MMP2/9 levels and activities. In clinical specimens of OSCCs, GIT1 levels were elevated relative to paired normal tissues and were correlated with lymph node metastasis, with expression levels of miR-491-5p and GIT1 correlated inversely in OSCCs, where they informed tumor grade. Together, our findings identify a functional axis for OSCC invasion that suggests miR-491-5p and GIT1 as biomarkers for prognosis in this cancer.

DSG3 facilitates cancer cell growth and invasion through the DSG3-plakoglobin-TCF/LEF-Myc/cyclin D1/MMP signaling pathway.

Desmoglein 3 (DSG3) is a component of the desmosome, which confers strong cell-cell adhesion. Previously, an oncogenic function of DSG3 has been found in head neck cancer (HNC). Here, we investigated how this molecule contributes to the malignant phenotype. Because DSG3 is associated with plakoglobin, we examined whether these phenotypic alterations were mediated through the plakoglobin molecule. Immunoprecipitation and immunofluorescence staining revealed that DSG3 silencing disrupted its interaction with plakoglobin and induced plakoglobin translocation from the cytoplasm to the nucleus. Knockdown of DSG3 significantly increased the interaction of plakoglobin with the transcriptional factor TCF and suppressed the TCF/LEF transcriptional activity. These effects further conferred to reduced expression of the TCF/LEF downstream target genes, including c-myc, cyclin D1, and MMP-7. Functional analyses showed that DSG3 silencing reduced cell growth and arrested cells at G0/G1 phase. Besides, cell migration and invasion abilities were also decreased. These cellular results were confirmed using tumor xenografts in mice, as DSG3 silencing led to the suppressed tumor growth, plakoglobin translocation and reduced expression of TCF/LEF target genes in tumors. Therefore, our study shows that the desmosomal protein DSG3 additionally functions to regulate malignant phenotypes via nuclear signaling. In conclusion, we found that DSG3 functions as an oncogene and facilitates cancer growth and invasion in HNC cells through the DSG3-plakoglobin-TCF/LEF pathway.

Oncogenic function and early detection potential of miRNA-10b in oral cancer as identified by microRNA profiling.

The miRNA participates in a variety of biologic processes, and dysregulation of miRNA is associated with malignant transformation. In this study, we determined specific profile of miRNA associated with oral cancer by using miRNA array screening method. There were 23 miRNAs found with considerably differential expressions between six oral cancer cell lines and five lines of normal oral keratinocytes, in which, 10 miRNAs showed the highest significant difference after independent examination by reverse transcription quantitative PCR. Eight molecules were upregulated, miR-10b, miR-196a, miR-196b, miR-582-5p, miR-15b, miR-301, miR-148b, and miR-128a, and two molecules, miR-503 and miR-31, were downregulated. The most upregulated miR-10b was further examined, and its functions were characterized in two oral cancer cell lines. The miR-10b actively promotes cell migration (2.6- to 3.6-fold) and invasion (1.7- to 1.9-fold) but has minimal effect on cell growth or chemo-/radiosensitivity. Furthermore, miR-10b was considerably elevated in the plasma of xenografted tumor mice (20-fold). This upregulation of miR-10b in plasma was further shown in the patients with oral cancer [P < 0.0001, area under curve (AUC) = 0.932] and precancer lesions (P < 0.0001, AUC = 0.967), suggesting that miR-10b possesses a high potential to discriminate the normal subjects. In conclusion, we have identified at least 10 miRNAs significantly associated with oral cancer, including the most elevated miR-10b. The miR-10b actively participates in cancer formation by promoting cell migration and invasion. Our study using clinical samples suggests that plasma miR-10b has high potential as an early detection marker for oral cancer.

Molecular chaperones as a common set of proteins that regulate the invasion phenotype of head and neck cancer.

PURPOSE: The goal of this study was to establish a common set of molecules that regulate cell invasion in head and neck cancer (HNC). EXPERIMENTAL DESIGN: Five invasive sublines derived from HNC cell lines were established using the Matrigel selection method. Proteomic technology, MetaCore algorithm, and reverse transcriptase-PCR methods were used to search for molecules that contribute to the invasion phenotype. Cellular functional analyses and clinical association studies were applied to examine the significance of the molecules. RESULTS: Fifty-two proteins were identified in more than two of the four independent proteomic experiments, including 10 (19%) molecular chaperones. Seven chaperones were confirmed to be differentially expressed in five sublines, Hsp90α, Hsp90ß, Hsp90-B1/Gp96, Hsp70-A5/Grp78, and HYOU1, that upregulate, whereas Hsp60 and glucosidase-α neutral AB (GANAB) downregulate. Four molecules were further investigated. In all cell lines, knockdown of Hsp60 or GANAB and silencing of Gp96 or Grp78 considerably enhanced or reduced cell migration and invasion, respectively. Clinical association studies consistently revealed that low levels of Hsp60 or GANAB and high levels of Gp96 or Grp78 are significantly associated with advanced cancer (P < 0.001 to P = 0.047, respectively, for the four molecules) and poor survival (P < 0.001 to P = 0.025, respectively, for the four molecules). CONCLUSION: Our study defined molecular chaperones as a common set of proteins that regulate the invasion phenotype of HNC. Loss of the tumor suppression function of Hsp60 or GANAB and acquisition of the oncogenic function of Gp96 or Grp78 contribute to aggressive cancers. These molecules may serve as prognostic markers and targets for cancer drug development.