Ya-Ching Lu

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.

hTERT phosphorylation by PKC is essential for telomerase holoprotein integrity and enzyme activity in head neck cancer cells

Telomerase activity is suppressed in normal somatic tissues but is activated in most cancer cells. We have previously found that all six telomerase subunit proteins, including hTERT and hsp90 are needed for full enzyme activity. Telomerase activity has been reported to be upregulated by protein kinase C (PKC), but the mechanism is not clear. In this study, we examined how PKC regulates telomerase activity in head and neck cancer cells. PKC inhibitor, bisindolylmaleimide I (BIS), inhibited telomerase activity but had no effect on the expressions of telomerase core subunits. RNA interference (RNAi) and in vitro phosphorylation studies revealed that PKC isoforms α, β, δ, ɛ, ζ specifically involved in telomerase regulation, and the phosphorylation target was on hTERT. Treatment with the hsp-90 inhibitor novobiocin dissociated hsp90 and hTERT as revealed by immunoprecipitation and immunoblot analysis and reduced telomerase activity. Treatment with the PKC activator SC-10 restored the association of hsp90 and hTERT and reactivate telomerase, suggesting that hTERT phosphorylation by PKC is essential for telomerase holoenzyme integrity and function. Analysis on clinical normal and tumour tissues reveal that the expressions of PKC α, β, δ, ɛ, ζ were higher in the tumour tissues, correlated with telomerase activity. Disruption of PKC phosphorylation by BIS significantly increased chemosensitivity to cisplatin. In conclusion, PKC isoenzymes α, β, δ, ɛ, ζ regulate telomerase activity in head and neck cancer cells by phosphorylating hTERT. This phosphorylation is essential for telomerase holoenzyme assembly, leading to telomerase activation and oncogenesis. Manipulation of telomerase activity by PKC inhibitors is worth exploring as an adjuvant therapeutic approach.

Highly potent and specific siRNAs against E6 or E7 genes of HPV16- or HPV18-infected cervical cancers

Infection with high-risk types (type 16 or type 18) of human papillomaviruses (HPVs) increases a patients risk of cervical cancer. Given the importance of the cervix and the severe side effects resulting from traditional cancer therapies, this study aimed to achieve targeted inhibition of viral oncogenes in tumor cells using small interfering RNAs (siRNA). To accomplish this, we developed nine siRNAs against either the E6 or E7 genes of HPV-16 or HPV-18 in several combinations, yielding siRNAs targeting 16E6, 16E7, 18E6 and 18E7. We measured the effectiveness of the siRNAs by examining E6 or E7 mRNA expression after transfection of the siRNAs into HPV-positive CaSki (HPV-16) or HeLa (HPV-18) cell lines. We found that the HPV-siRNAs significantly reduced cell growth and colony formation in both cell lines. Flow cytometry analysis revealed a significant increase in apoptosis. The siRNAs had no effect on cell growth, colony formation or apoptosis in HPV-negative C33A cells, demonstrating a lack of off-target effects. In addition, an in vivo xenograft study showed that intra-tumoral injection of the siRNAs reduced tumor growth in BALB/c nude mice. In conclusion, we have developed highly specific and potent HPV-siRNAs that successfully suppress tumor growth and induce apoptosis in HPV-positive cervical cancer cells. siRNA treatment has potential for further development as an adjuvant therapy for cervical cancer.

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

BackgroundMicroRNA-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.MethodsCellular 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.ResultsBoth 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 its their function by inhibiting NME4 expression and further activating p-JNK, suppressing TIMP1, and augmenting MMP1/9.ConclusionmiR-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.

Grp78 as a therapeutic target for refractory head–neck cancer with CD24 − CD44 + stemness phenotype

Cancer stem cells are refractory to conventional therapy, which result to cancer metastasis and chemo-radioresistance. Grp78 is known to have important roles in cytoprotection and tumorigenesis in several cancers. We therefore examined whether Grp78 can serve as a therapeutic target for refractory stemness phenotype of head and neck cancer (HNC). Six HNC cell lines were used. Fluorescence-activated cell sorting (FACS) analysis was used to sort CD24−CD44+ and Grp78+ cells. The small interfering RNA (siRNA) knockdown and cDNA transfection were applied to examine the effects of Grp78 on cellular function. Western blot and confocol microscopy were used to determine the effects of downstream protein expressions. Xenografted mouse tumors and immunohistochemistry were used to validate the results. We found that Grp78 regulated the conversion of CD24−CD44+ cells, a characteristic of HNC stem cells. The CD24−CD44+Grp78+ cells showed superior chemo-radioresistance and invasion ability compared with CD24−CD44+, Grp78+ or the parental cells. Silencing Grp78 increased chemo-radiosensitivity, inhibited cell invasion, reverse epithelial–mesenchymal transition, suppressed cancer stemness, withdrew CD24−CD44+ cell conversion and induced differentiated phenotype. Study in xenografted mice further showed that CD24−CD44+Grp78+ cells exhibited highest tumorigenesis, compared with CD24−CD44+ CD24+CD44+ or the parental cells. Grp78 knockdown dramatically restrained tumor growth along with the inhibition of stem cell regulatory proteins Oct-4 and Slug. Grp78 may serve as a molecular target that can be further developed for eradication of refractory HNC with stemness phenotype.

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.

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.

Abstract 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.

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-β 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.

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.