Ming Yung Chou

Markedly increased Oct4 and Nanog expression correlates with cisplatin resistance in oral squamous cell carcinoma

BACKGROUND Oral squamous cell carcinoma (OSCC) is the sixth most prevalent cancer worldwide. Cancer stem cells (CSC) model theoretically contribute to tumor growth, metastasis, and chemo-radioresistance. Cisplatin is a widely used chemotherapeutic agent for OSCC treatment. The aim of this study was to compare stemness genes expression in chemo-sensitive and chemo-resistant specimens and further explore the potential markers that may lead to induce chemo-resistance in OSCC. METHODS The study method is the treatment of OC2 cells with cisplatin select cisplatin-resistant OC2 cells. Self-renewal ability was evaluated by cultivating parental and cisplatin-resistant OC2 cells within sphere-forming assay after serial passages. Differential expression profile of stemness markers between parental and cisplatin-resistant OC2 cells was elucidated. The parental and cisplatin-resistant OC2 cells were assessed for migration/invasion/clonogenicity tumorigenic properties in vitro. Expression of stemness markers in chemo-sensitive and chemo-resistant patients with OSCC was performed by immunohistochemistry staining in vivo. RESULTS Sphere-forming/self-renewal capability was increased in cisplatin-resistant OC2 cells. Cisplatin-resistant OC2 cells highly expressed the stemness markers (Nanog, Oct4, Bmi1, CD117, CD133, and ABCG2). Furthermore, cisplatin-resistant OC2 cells increased migration/invasion/clonogenicity ability. Notably, up-regulation of Oct4 and Nanog expression was significantly observed in cisplatin-resistant patients with OSCC (**P < 0.01). CONCLUSIONS These data indicate that cancer stem-like properties were expanded during the acquisition of cisplatin resistance in OSCC. Clinically, oral cancer stemness markers (Oct4 and Nanog) overexpression may promote the OSCCs recurrence to resist cisplatin.

Let-7d functions as novel regulator of epithelial-mesenchymal transition and chemoresistant property in oral cancer

Oral squamous cell carcinoma (OSCC) is a prevalent cancer worldwide. Let-7 family has been shown to function as a tumor suppressor through regulating multiple oncogenic signaling. Recent study reported that combined underexpression of miR-205 and let-7d showed negative correlation with the survival prognosis of head and neck cancer patients. However, the let-7d-involved mechanism in regulating OSCC is still unclear. In this study, we first demonstrated that let-7d expression was significantly decreased while Twist and Snail expression was increased in OSCC cancer cell lines and primary cultures as compared to normal human oral keratinocyte cells. To further investigate the role of let-7d in OSCC, we applied the SPONGE method to knock down let-7d in OECM-1 and two primary OSCC cell types. The results showed that knockdown of let-7d promote epithelial-mesenchymal transition (EMT) traits and migratory/invasive capabilities in OSCC cells. Furthermore, down-expression of let-7d significantly activated Twist and Snail expressions and chemo-resistant abilities of OSCC cells. Notably, overexpression of let-7d effectively reversed the EMT phenotype, blocked migratory/invasive abilities, and further increased the chemosensitivity in oral cancer tumor initiating ALDH1+ cells. In sum, these results show that let-7d negatively modulates EMT expression and also plays a role in regulating chemo-resistant ability in oral cancer.

MicroRNA let-7a represses chemoresistance and tumourigenicity in head and neck cancer via stem-like properties ablation

Head and neck cancer (HNC) is a prevalent cancer worldwide. Let-7 has been shown to function as a tumour suppressor by regulating multiple oncogenic signalling pathways. However, the role of let-7 in head and neck cancer (HNC) and in HNC-associated tumour initiating cells (TIC) remains unclear. In this study, we first demonstrated that let-7a expression was significantly decreased but that Nanog/Oct4 expression was increased in HNC tissues as compared to adjacent normal cells. Expression of let-7a in recurrent HNC tissue and in regional metastatic lymph nodes of HNC patients was also significantly decreased, but Nanog/Oct4 expression was increased as compared to the expression levels in the parental tumours. Consistently, the stemness genes were significantly up-regulated and let-7a was down-regulated in HNC-ALDH1(+) cells relative to HNC-ALDH1(-) cells. Furthermore, lentiviral-mediated let-7a overexpression could significantly inhibit the stemness signature and the chemoresistant abilities of HNC-ALDH1(+) cells. Most importantly, overexpression of let-7 or knockdown of Nanog in ALDH1(+) cells effectively blocked tumour metastasis and significantly prolonged survival time in ALDH1(+)-transplanted immunocompromised mice. Overall, restoration of let-7a in HNC and HNC-TIC may be a new approach for the therapeutic treatment of HNC in the future. These results show that let-7a negatively modulates the expression of stemness genes and plays a role as a tumour suppressor in HNC by eliminating the putative HNC-TIC population.

miR145 Targets the SOX9/ADAM17 Axis to Inhibit Tumor-Initiating Cells and IL-6–Mediated Paracrine Effects in Head and Neck Cancer

ALDH1(+)CD44(+) cells are putative tumor-initiating cells (TIC) in head and neck squamous cell carcinomas (HNC). miR-145 regulates tumorigenicity in various cancers but the breadth of its mechanistic contributions and potential therapeutic applications are not completely known. Here, we report that ALDH1(+)CD44(+)-HNC cells express reduced levels of miR145. SPONGE-mediated inhibition of miR-145 (Spg-miR145) was sufficient to drive tumor-initiating characteristics in non-TICs/ALDH1(-)CD44-negative HNC cells. Mechanistic analyses identified SOX9 and ADAM17 as two novel miR145 targets relevant to this process. miR-145 expression repressed TICs in HNC in a manner associated with SOX9 interaction with the ADAM17 promoter, thereby activating ADAM17 expression. Notably, the SOX9/ADAM17 axis dominated the TIC-inducing activity of miR-145. Either miR-145 suppression or ADAM17 overexpression in non-TICs/ALDH1(-)CD44(-)-HNC cells increased expression and secretion of interleukin (IL)-6 and soluble-IL-6 receptor (sIL-6R). Conversely, conditioned medium from Spg-miR145-transfected non-TICs/ALDH1(-)CD44(-)-HNC cells was sufficient to confer tumor-initiating properties in non-TICs/ALDH1(-)CD44(-)-HNC and this effect could be abrogated by an IL-6-neutralizing antibody. We found that curcumin administration increased miR-145 promoter activity, thereby decreasing SOX9/ADAM17 expression and eliminating TICs in HNC cell populations. Delivery of lentivral-miR145 or orally administered curcumin blocked tumor progression in HNC-TICs in murine xenotransplant assays. Finally, immunohistochemical analyses of patient specimens confirmed that an miR-145(low)/SOX9(high)/ADAM17(high) phenotype correlated with poor survival. Collectively, our results show how miR-145 targets the SOX9/ADAM17 axis to regulate TIC properties in HNC, and how altering this pathway may partly explain the anticancer effects of curcumin. By inhibiting IL-6 and sIL-6R as downstream effector cytokines in this pathway, miR-145 seems to suppress a paracrine signaling pathway in the tumor microenvironment that is vital to maintain TICs in HNC.

Impairment of tumor‐initiating stem‐like property and reversal of epithelial–mesenchymal transdifferentiation in head and neck cancer by resveratrol treatment

SCOPE Recent reports have demonstrated that head and neck cancer-derived tumor-initiating cells (HNC-TICs) presented high tumorigenic, chemoradioresistant, metastatic properties, and were coupled with gain of epithelial-mesenchymal transition (EMT) characteristics. The aim of this study was to investigate the chemotherapeutic effect and regulatory mechanisms of resveratrol on HNC-TICs. METHODS AND RESULTS We first observed that the treatment of resveratrol significantly downregulated the ALDH1 activity and CD44 positivity of head and neck cancer (HNC) cells in a dose-dependent manner (p < 0.05). Moreover, resveratrol treatment reduced self-renewal property and stemness genes signatures (Oct4, Nanog, and Nestin) expression in sphere-forming HNC-TICs. Additionally, the repressive effect of resveratrol on in vitro malignant properties including invasiveness/anchorage-independent growth was mediated by regulating productions of EMT markers Slug, ZEB1, N-cadherin, E-cadherin, and Vimentin. Importantly, an in vivo nude mice model showed that resveratrol treatment to xenograft tumors by oral gavage reduced tumor growth, stemness, and EMT markers in vivo. Lastly, synergistic effect of resveratrol and conventional chemotreatment attenuated tumor-initiating cells property in HNC-TICs. CONCLUSIONS Our results demonstrated that resveratrol would be a valuable therapeutics clinically in combination with conventional chemotherapy treatment modalities for malignant HNCs by elimination of tumor-initiating stem-like and EMT properties.

Suppression of miR-204 enables oral squamous cell carcinomas to promote cancer stemness, EMT traits, and lymph node metastasis.

The feature of oral squamous cell carcinomas (OSCC) is commonly metastasizing to locoreginal lymph nodes, and the involvement of lymph nodes metastasis represents the one of important prognostic factors of poor clinical outcome. MicroRNAs (miRNAs) have been shown to be key players of cancer-related hallmarks including cancer stemness, EMT (epithelial-mesenchymal transition), and metastaisis. Herein we showed that OSCC-derived ALDH1+ cancer stem cells (OSCC-CSCs) express lower level of miR-204, and miR-204 over-expression suppresses cancer stemness and in vivo tumor-growth of OSCC-CSCs. miR-204 binds on their 3′UTR-regions of Slug and Sox4 and suppressing their expression in OSCC-CSCs. On the contrary, down-regulation of miR-204 significantly increased cancer stemness and the lymph nodes incidence of orthotopic animal models. Furthermore, co-knockdown with sh-Slug and sh-Sox4 synergistically rescued miR-204-supressing cancer stemness and EMT properties. Clinical results further revealed that a miR-204lowSlughighSox4high signature predicted the worse survival prognosis of OSCC patients by Kaplan-Meier survival analyses. Up-regulated miR-204-targeting Slug and Sox4 by epigallocatechin-3-gallate (EGCG) treatment significantly inhibited the proliferation rate, self-renewal capacity, and the percentage of ALDH1+ and CD44+ cells in OSCC-CSCs Oral-feeding of EGCG effectively alleviated tumor-progression in OSCC-CSCs-xenotransplanted immunocompromised mice through miR-204 activation. In conclusion, miR-204-mediated suppression of cancer stemness and EMT properties could be partially augmented by the anti-CSCs effect of EGCG.

Concurrent Expression of Oct4 and Nanog Maintains Mesenchymal Stem-Like Property of Human Dental Pulp Cells

Human dental pulp stem cells (DPSCs), unique mesenchymal stem cells (MSCs) type, exhibit the characteristics of self-renewal and multi-lineage differentiation capacity. Oct4 and Nanog are pluripotent genes. The aim of this study was to determine the physiological functions of Oct4 and Nanog expression in DPSCs. Herein, we determined the critical role of an Oct4/Nanog axis modulating MSCs properties of DPSCs by lentiviral-mediated co-overexpression or co-knockdown of Oct4/Nanog in DPSCs. MSCs properties including osteogenic/chondrogenic/adipogenic induction differentiation was assayed for expression of osteogenic/chondrogenic/adipogenic markers by quantitative real-time RT-PCR analysis. Initially, we observed that the expression profile of Oct4 and Nanog in dental pulp cells, which exerted properties of MSCs, was significantly up-regulated compared to that of STRO-1−CD146− dental pulp cells. Down-regulation of Oct4 and Nanog co-expression significantly reduced the cell proliferation, osteogenic differentiation capability, STRO-1, CD146, and Alkaline phosphatase (ALP) activity of DPSCs. In contrast, co-overexpression of Oct4 and Nanog enhanced the expression level of STRO-1 and CD146, proliferation rate and osteogenic/chondrogenic/adipogenic induction differentiation capability, and expression of osteogenic/chondrogenic/adipogenic induction differentiation markers. Our results suggest that Oct4-Nanog signaling is a regulatory switch to maintain properties in DPSCs.

Enhanced Chemosensitivity by Targeting Nanog in Head and Neck Squamous Cell Carcinomas

Chemo-resistance is the major cause of high mortality in head and neck squamous cell carcinomas (HNSCC) in which HNSCC-derived cancer stem cells (CSCs) may be involved. Previously, we enriched a subpopulation of HNSCC-derived spheroid cells (SC) (HNSCC-SC) and identified Nanog as a CSCs marker. The aim of this study was to determine the role of Nanog in the chemosensitivity of HNSCC. The functional and clinicopathological studies of Nanog were investigated in HNSCC cells and specimens. Nanog expression was increased in HNSCC cell lines as compared to a normal oral epithelial cell line. Nanog upregulation in clinical tissues from HNSCC patients with recurrent and metastatic specimens relative to the mRNA levels in the samples from normal or primary tissues were examined. Targeting Nanog in HNSCC-SC significantly inhibited their tumorigenic and CSCs-like abilities and effectively increased the sensitivity of HNSCC-SC to chemotherapeutic drug cisplatin treatment. Targeting Nanog in HNSCC-SC showed a synergistic therapeutic effect with cisplatin. Our results suggest that targeting Nanog may have promising therapeutic potential for HNSCC.

β-catenin expression in areca quid chewing-associated oral squamous cell carcinomas and upregulated by arecoline in human oral epithelial cells

BACKGROUND/PURPOSE Nuclear localization of β-catenin is known to associate with malignant transformation of many squamous cell carcinomas. The aim of this study was to compare β-catenin expression in normal human oral epithelium and areca quid chewing associated oral squamous cell carcinomas (OSCCs) and further to explore the potential mechanisms that may lead to induce β-catenin expression. METHODS A total of 40 areca quid chewing-associated OSCCs and 10 normal oral tissue biopsy samples without areca quid chewing were analyzed by immunohistochemistry. The oral epithelial cell line GNM cells were challenged with arecoline, a major areca nut alkaloid, by using Western blot analysis. Furthermore, extracellular signal-regulated protein kinase inhibitor PD98059, glutathione precursor N-acetyl-l-cysteine (NAC), tyrosine kinase inhibitor herbimycin-A, p38 inhibitor SB203580, and phosphatidylinositaol 3-kinase inhibitor LY294002 were added to find the possible regulatory mechanisms. RESULTS β-catenin expression was significantly higher in OSCC specimens than that in normal oral epithelial specimens (p < 0.05). It was demonstrated that normal oral epithelium showed only membranous staining for β-catenin, and membranous staining was lost or reduced with an increase in cytoplasmic/nuclear staining in OSCCs. Arecoline was found to elevate β-catenin expression in a dose-dependent manner (p < 0.05). The addition of PD98059, NAC, herbimycin-A, SB203580, and LY294002 markedly inhibited the arecoline-induced β-catenin expression (p < 0.05). CONCLUSION β-catenin expression is significantly upregulated in areca quid chewing-associated OSCC. The localization of β-catenin expression is correlated with the tumor size and clinical stage. In addition, β-catenin expression induced by arecoline is downregulated by PD98059, NAC, herbimycin-A, SB203580, and LY294002.

Expression of carbonic anhydrases I/II and the correlation to clinical aspects of oral squamous cell carcinoma analyzed using tissue microarray

BACKGROUND Carbonic anhydrases (CA), a family of metalloenzymes, play an important role in catalyzing the equilibration of carbon dioxide (CO(2)) and carbonic acid (H(2)CO(3)). The role of CAs in tumorigenesis is controversial, especially regarding the expression of CA isoenzymes between various tumor types. This study explores the correlation between the expressions of CA I and CA II and the characteristic features of oral cancer. METHODS We immunohistochemically examined the expressions of CA I and CA II in 279 cases of oral squamous cell carcinoma (OSCC) using tissue microarrays. Additionally, the oral cancer cell line SCC-9 was used to confirm the relationship between CA I and CA II expression and cell growth. RESULTS We found a significant correlation between positive CA I and CA II stains and OSCC for more advanced clinical stage (P = 0.014 or 0.012) and larger tumor size (P = 0.008 or 0.038), but not for positive lymph node metastasis, distal metastasis, and recurrence. In vitro analysis also showed that treatment with a CA inhibitor, acetazolamide, inhibited the growth of SCC-9 cells. CONCLUSION We conclude that expressions of CA I and CA II in OSCC samples can be used to predict local tumor growth in OSCC patients.