Shou-Yen Kao

Bmi1 is essential in Twist1-induced epithelial-mesenchymal transition

The epithelial–mesenchymal transition (EMT), one of the main mechanisms underlying development of cancer metastasis, induces stem-like properties in epithelial cells. Bmi1 is a polycomb-group protein that maintains self-renewal, and is frequently overexpressed in human cancers. Here, we show the direct regulation of BMI1 by the EMT regulator, Twist1. Furthermore, Twist1 and Bmi1 were mutually essential to promote EMT and tumour-initiating capability. Twist1 and Bmi1 act cooperatively to repress expression of both E-cadherin and p16INK4a. In patients with head and neck cancers, increased levels of both Twist1 and Bmi1 correlated with downregulation of E-cadherin and p16INK4a, and was associated with the worst prognosis. These results suggest that Twist1-induced EMT and tumour-initiating capability in cancer cells occurs through chromatin remodelling, which leads to unfavourable clinical outcomes.

Increase of microRNA miR-31 level in plasma could be a potential marker of oral cancer

BACKGROUNDS Oral squamous cell carcinoma (OSCC) is a worldwide disease. MicroRNAs are endogenously expressed non-coding RNAs that have important biological and pathological functions. miR-31 was found markedly up-regulated in OSCC and several other malignancies. However, miR-31 expression was also down-regulated in the metastasis process of breast carcinoma. MATERIALS AND METHODS Using quantitative RT-PCR analysis, we identified plasma miR-31 in OSCC patients (n = 43) and case controlled individuals (n = 21). Nine OSCC patients saliva were also analyzed. The Mann-Whitney test and Wilcoxon matched pairs test were used to compare the differences among the various clinical variants. RESULTS miR-31 in plasma was significantly elevated in OSCC patients relative to age and sex-matched control individuals. This marker yielded a receiver operating characteristic curve area of 0.82 and an accuracy of 0.72 defined by leave-one-out cross-validation. In addition, the plasma miR-31 in patients was remarkably reduced after tumor resection suggesting that this marker is tumor associated. Our preliminary analysis also demonstrated the feasibility of detecting the increase of miR-31 in patients saliva. CONCLUSION This study concluded that plasma miR-31 could be validated a marker of OSCC for diagnostic uses.

Outcomes of oral squamous cell carcinoma in Taiwan after surgical therapy: factors affecting survival.

PURPOSE The study goal was to determine which clinical features correlated with 5-year survival in patients surgically treated for oral squamous cell carcinoma (OSCC) in Taiwan. PATIENTS AND METHODS The records of 378 OSCC patients surgically treated with or without chemotherapy and radiotherapy were reviewed retrospectively. Their 5-year survival in relation to age, gender, tumor site, lymph node involvement, presence of distant metastasis, staging, differentiation, and risk factors, including betel quid (BQ) chewing, cigarette smoking, and alcohol consumption, was analyzed. RESULTS The majority of the patients were men (male-to-female ratio, 5.87:1) with the mean age of 57.1 +/-11.7 years. Tumors occurred mainly at the buccal mucosa (BM) (100 of 378, 26.5%), gingiva (105 of 378, 27.8%), and tongue (103 of 378, 27.2%). Neck nodal metastasis occurred frequently at the floor of the mouth (in >60% of cases), followed by the gingiva (45.7%), buccal mucosa (34%), and tongue (20.4%), whereas early distant metastasis was rare (5.3%). There were 104 (27.5%) stage 1, 96 (25.4%) stage 2, 98 (25.9%) stage 3, and 80 (21.2%) stage 4 patients. OSCC at the BM and gingiva was most (and at the tongue least) associated with risk factors of BQ use and smoking. The 5-year survival was 75%, 65.6%, 49%, and 30% for patients with stage I, II, III, and IV, respectively. The size, nodal involvement, distant metastasis, staging, differentiation, and BQ use significantly affected the survival (P <.05, Kaplan-Meier analysis). BQ use also correlated most significantly with the younger age of occurrence of OSCC patients. CONCLUSIONS Our data suggest that early treatment is the key to increasing the survival of OSCC patients. Periodic screening of high-risk populations for OSCC represents an urgent need in Taiwan.

RAC1 activation mediates Twist1-induced cancer cell migration.

Epithelial–mesenchymal transition (EMT), which is characterized by the suppression of the adhesion protein E-cadherin, is a crucial process that promotes metastasis and stem-like properties of cancer cells. However, the dissociation of cellular aggregates is not sufficient to explain why cancer cells move, and the motile nature of cancer cells undergoing EMT remains elusive. Here, we identify a mechanism in which the EMT inducer Twist1 elicits cancer cell movement through activation of RAC1. Twist1 cooperates with BMI1 to suppress let-7i expression, which results in upregulation of NEDD9 and DOCK3, leading to RAC1 activation and enabling mesenchymal-mode movement in three-dimensional environments. Moreover, the suppression of let-7i contributes to Twist1-induced stem-like properties. Clinically, activation of the Twist1–let-7i–NEDD9 axis in head and neck cancer patients correlates with tumour invasiveness and worse outcome. Our results uncover an essential mechanism to explain how Twist1 induces the motile stem-like cancer cell phenotype beyond simply suppressing E-cadherin.

MicroRNA-200c attenuates tumour growth and metastasis of presumptive head and neck squamous cell carcinoma stem cells†

MicroRNA‐200c (miR200c) is emerging as an important regulator of tumourigenicity and cancer metastasis with a strong capacity for inducing epithelial–mesenchymal transitions. However, the role of miR200c in head and neck squamous cell carcinoma (HNSCC) and HNSCC‐associated cancer stem cells (HNSCC‐CSCs) is unknown. In this study, the expression of miR200c in the regional metastatic lymph node of HNSCC tissues was significantly decreased, but BMI1 expression was increased as compared to parental tumours. Importantly, site‐directed mutagenesis with a luciferase reporter assay showed that miR200c targeted the 3′ UTR of BMI1 in HNSCC cells. Isolated HNSCC‐derived ALDH1+/CD44+ cells displayed CSC‐like tumour initiating and radio‐resistant properties. The expression levels of miR200c were significantly down‐regulated while BMI1 was increased in HNSCC‐ALDH1+/CD44+ compared to the other subsets of HNSCC cells. Furthermore, increased miR200c expression or knockdown of BMI1 could significantly inhibit the malignant CSC‐like properties of ALDH1+/CD44+ cells. miR200c over‐expression further down‐regulated the expressions of ZEB1, Snail and N‐cadherin, but up‐regulated E‐cadherin expression in ALDH1+/CD44+ cells. Finally, a xenotransplantion study confirmed that over‐expression of miR200c or BMI1 knockdown effectively inhibited the lung metastatic ability and prolonged the survival rate of ALDH1+/CD44+‐transplanted mice. In summary, miR200c negatively modulates the expression of BMI1 but also significantly inhibits the metastatic capability of epithelial–mesenchymal transitions in malignant HNSCC by reducing the expression of BMI1/ZEB1. Restoration of miR200c in HNSCC and CSCs may be a promising therapeutic approach. Copyright

Inhibition of tumorigenicity and enhancement of radiochemosensitivity in head and neck squamous cell cancer-derived ALDH1-positive cells by knockdown of Bmi-1

Bmi-1, a member of the Polycomb family of transcriptional repressors, is essential for maintaining the self-renewal abilities of adult stem cells. Bmi-1 has been demonstrated to play a role in tumorigenesis in head and neck squamous cell carcinomas (HNSCCs). A recent study has further suggested that ALDH1 may be considered to be a putative marker for HNSCC-derived cancer stem cells. However, the role that Bmi-1 plays in HNSCC-derived ALDH1-positive cells (HNSCC-ALDH1(+)) has yet to be determined. In this study, we demonstrated that HNSCC-ALDH1(+) cells possess tumor initiating properties, are capable of self-renewal, and express higher levels of Bmi-1 as compared to HNSCC-ALDH1(-) cells. To further explore the functional role of Bmi-1 in HNSCC-ALDH1(+) cells, we used a lentiviral vector expressing shRNA to knock down Bmi-1 expression (sh-Bmi-1) in HNSCC-ALDH1(+) cells. Silencing of Bmi-1 significantly enhanced the sensitivity of HNSCC-ALDH1(+) cells to chemoradiation and increased the degree of chemoradiation-mediated apoptosis that occurred. Importantly, knockdown of Bmi-1 increased the effectiveness of radiotherapy and led to the inhibition of tumor growth in nude mice transplanted with HNSCC-ALDH1(+) cells. Kaplan-Meier survival analysis indicated that the mean survival rate of HNSCC-ALDH1(+) tumor-bearing immunocompromised mice treated with radiotherapy was significantly improved by treatment with sh-Bmi-1 as well. In summary, these results suggest that Bmi-1 is a potential target for increasing the sensitivity of HNSCC cancer stem cells to chemoradiotherapy.

Bmi-1 Regulates Snail Expression and Promotes Metastasis Ability in Head and Neck Squamous Cancer-Derived ALDH1 Positive Cells

Recent studies suggest that ALDH1 is a putative marker for HNSCC-derived cancer stem cells. However, the regulation mechanisms that maintain the stemness and metastatic capability of HNSCC-ALDH1+ cells remain unclear. Initially, HNSCC-ALDH1+ cells from HNSCC patient showed cancer stemness properties, and high expression of Bmi1 and Snail. Functionally, tumorigenic properties of HNSCC-ALDH1+ cells could be downregulated by knockdown of Bmi-1. Overexpression of Bmi-1 altered in expression property ALDH1− cells to that of ALDH1+ cells. Furthermore, knockdown of Bmi-1 enhanced the radiosensitivity of radiation-treated HNSCC-ALDH1+ cells. Moreover, overexpression of Bmi-1 in HNSCC-ALDH1− cells increased tumor volume and number of pulmonary metastatic lesions by xenotransplant assay. Importantly, knock-down of Bmi1 in HNSCC-ALDH1+ cells significantly decreased distant metastases in the lungs. Clinically, coexpression of Bmi-1/Snail/ALDH1 predicted the worst prognosis in HNSCC patients. Collectively, our data suggested that Bmi-1 plays a key role in regulating Snail expression and cancer stemness properties of HNSCC-ALDH1+ cells.

Accumulation of mitochondrial DNA deletions in human oral tissues — effects of betel quid chewing and oral cancer

Accumulation of mitochondrial DNA (mtDNA) mutations in human tissues has been associated with intrinsic aging and environmental insult. Recently, mtDNA mutations have been detected in various tumors, including head and neck tumors. However, the factors affecting the occurrence and accumulation of mtDNA deletions in tumor tissues are poorly understood. In Taiwan, betel quid chewing is a major risk factor for oral cancer. Using polymerase chain reaction (PCR) techniques, we examined large-scale deletions of mtDNA in 53 pairs of tumor and non-tumor oral tissues from the patients with or without betel quid chewing history. The results revealed that irrespective of the history of betel quid chewing, the incidences of the 4977bp deletion and other deletions of mtDNA were lower in the tumor portion as compared with the non-tumor portion. The average proportions of the 4977bp deleted mtDNA in the tumor tissues of the betel quid chewers and non-betel quid chewers were 13- and 5-fold, respectively, lower than those in the corresponding non-tumor tissues. Moreover, the average proportion of 4977bp deleted mtDNA was significantly higher (P<0.05) in the non-tumor oral tissues of the patients with betel quid chewing history than that of the patients without the history of betel quid chewing. These results suggest that betel quid chewing may increase mtDNA mutation in human oral tissues and that accumulation of mtDNA deletions and subsequent cytoplasmic segregation of these mutations during cell division could be an important contributor to the early phase of oral carcinogenesis.

miR-134 induces oncogenicity and metastasis in head and neck carcinoma through targeting WWOX gene.

Head and neck squamous cell carcinoma (HNSCC) is a prevalent disease worldwide, and the survival of HNSCC has not improved significantly over the last few decades. MicroRNAs (miRNAs) have an important regulatory role during carcinogenesis. Our study investigated the pathogenic implications of miR‐134 in head and neck carcinogenesis. The clinicopathologic implications of miR‐134 in HNSCC were investigated using expression assays and the functional role of miR‐134 in HNSCC pathogenesis was determined using ectopic expression, knockdown and reporter assay experiments. Xenographic tumorigenesis and orthotopic nodal metastasis were assayed in mouse models. In situ hybridization and immunohistochemistry were used to detect the expression of miR‐134 and the WWOX gene in human HNSCC. The results indicated that miR‐134 was upregulated in HNSCC tissues relative to control mucosa. High expression of miR‐134 was associated with nodal metastasis and mortality of patients. Decreased plasma miR‐134 levels after tumor ablation indicated a better prognosis for patients. Multivariate analysis showed that high miR‐134 expression in HNSCC was an independent predictor of poor survival. Ectopic miR‐134 expression significantly enhanced in vitro oncogenic phenotypes and the orthotopic growth and metastasis of HNSCC cells. miR‐134 targeted WW domain‐containing oxidoreductase (WWOX) gene and cell invasion enhanced by miR‐134 expression was abrogated by ectopic WWOX expression in HNSCC cells. miR‐134 expression was reversely associated with the WWOX expression in HNSCC tissues. Evidences from our study substantiated that miR‐134 expression contributes to head and neck carcinogenesis by targeting the WWOX.

Type I collagen promotes proliferation and osteogenesis of human mesenchymal stem cells via activation of ERK and Akt pathways

Biomaterials not only serve as scaffolds for bone regeneration, but may also exhibit inductive capability for bone growth. The goal of this study was to identify the best extracellular matrix protein for enhancing osteogenesis by hMSCs (human mesenchymal stem cells) and to investigate the underlying mechanism. Coating with collagen I, but not fibronectin, laminin, gelatin, and poly-L-lysine, enhanced late cell proliferation and promoted osteogenesis by hMSCs, as evidenced by an increase in Alizarin Red S staining, alkaline phosphatase activity and mRNA levels of Runx2 and osteocalcin. Coating with collagen I induced activation of ERK and Akt but not FAK, and treatment with PD98059 and LY294002 blocked the activation of ERK and Akt, respectively. Interestingly, LY294002 also blocked ERK activation, indicating the activation of PI3K/ERK pathway upon contact with collagen I. Furthermore, PD98059 or LY294002 abolished collagen I-induced promotion of osteogenesis by hMSCs. However, blocking antibodies against alpha2beta1 integrins did not inhibit collagen I-induced osteogenesis by hMSCs. These data demonstrate that collagen I promotes proliferation and osteogenesis of hMSCs via activation of ERK and Akt pathways.