Cheng-Chieh Yang

Tyrosine phosphorylation controls PCNA function through protein stability.

The proliferating cell nuclear antigen (PCNA) is an essential protein for DNA replication and damage repair. How its function is controlled remains an important question. Here, we show that the chromatin-bound PCNA protein is phosphorylated on Tyr 211, which is required for maintaining its function on chromatin and is dependent on the tyrosine kinase activity of EGF receptor (EGFR) in the nucleus. Phosphorylation on Tyr 211 by EGFR stabilizes chromatin-bound PCNA protein and associated functions. Consistently, increased PCNA Tyr 211 phosphorylation coincides with pronounced cell proliferation, and is better correlated with poor survival of breast cancer patients, as well as nuclear EGFR in tumours, than is the total PCNA level. These results identify a novel nuclear mechanism linking tyrosine kinase receptor function with the regulation of the PCNA sliding clamp.

CDK1-dependent phosphorylation of EZH2 suppresses methylation of H3K27 and promotes osteogenic differentiation of human mesenchymal stem cells.

Enhancer of zeste homologue 2 (EZH2) is the catalytic subunit of Polycomb repressive complex 2 (PRC2) and catalyses the trimethylation of histone H3 on Lys 27 (H3K27), which represses gene transcription. EZH2 enhances cancer-cell invasiveness and regulates stem cell differentiation. Here, we demonstrate that EZH2 can be phosphorylated at Thr 487 through activation of cyclin-dependent kinase 1 (CDK1). The phosphorylation of EZH2 at Thr 487 disrupted EZH2 binding with the other PRC2 components SUZ12 and EED, and thereby inhibited EZH2 methyltransferase activity, resulting in inhibition of cancer-cell invasion. In human mesenchymal stem cells, activation of CDK1 promoted mesenchymal stem cell differentiation into osteoblasts through phosphorylation of EZH2 at Thr 487. These findings define a signalling link between CDK1 and EZH2 that may have an important role in diverse biological processes, including cancer-cell invasion and osteogenic differentiation of mesenchymal stem cells.

Exploiting salivary miR-31 as a clinical biomarker of oral squamous cell carcinoma

Oral carcinoma is an important malignancy throughout the world. MicroRNAs (miRNAs) are endogenously expressed, non‐coding RNAs that regulate post‐transcriptional levels of targeted mRNAs. MiRNA‐31(miR‐31) is significantly upregulated in oral carcinoma tissues and plays oncogenic roles in oral carcinogenesis.

miR-181 as a putative biomarker for lymph-node metastasis of oral squamous cell carcinoma.

BACKGROUND Oral squamous cell carcinoma (OSCC) is an important malignant disease around the world. Aberrant expression of MicroRNAs (miRNAs) has been implicated in carcinogenesis of various cancers. In previous studies, up-regulation of miR-181 was observed when OSCC progressed from leukoplakia, dysplasia to invasive carcinoma. However, the function of miR-181 in oral tumorigenesis remains unclear. MATERIALS AND METHODS The expression levels of miR-181 in the tissue and plasma of OSCC patients were measured by quantitative RT-PCR. The correlation between miR-181 level and multiple clinical variables were then checked by Mann-Whitney test and Wilcoxon matched pairs test. To study the functional meaning of up-regulated miR-181, migration assay and invasion assay by transwells and colony forming assay were applied to analyze the tumorigenic phenotypes of OSCC cells with ectopical expression of miR-181. RESULTS Among different clinical variables, over-expression of miR-181 was correlated with lymph-node metastasis, vascular invasion, and a poor survival. Functional assays revealed ectopically over-expressed miR-181 would enhance cell migration and invasion, but not the ability of anchorage-independent growth of OSCC cells. In addition, the up-regulation of miR-181 could be detected both in tumor tissues and plasma. CONCLUSION miR-181 may enhance lymph-node metastasis through regulating migration, which could potentially be exploited as a putative biomarker for patients with OSCC.

miR-146a Enhances the Oncogenicity of Oral Carcinoma by Concomitant Targeting of the IRAK1, TRAF6 and NUMB Genes

MicroRNAs are short non-coding RNAs that regulate gene expression and are crucial to tumorigenesis. Oral squamous cell carcinoma (OSCC) is a prevalent malignancy worldwide. Up-regulation of miR-146 has been identified in OSCC tissues. However, the roles of miR-146 in carcinogenesis are controversial as it is suppressive in many other malignancies. The present study investigated the pathogenic implications of miR-146a in oral carcinogenesis. Microdissected OSCC exhibits higher levels of miR-146a expression than matched adjacent mucosal cells. The plasma miR-146a levels of patients are significantly higher than those of control subjects; these levels decrease drastically after tumor resection. miR-146a levels in tumors and in patients’ plasma can be used to classify OSCC and non-disease status (sensitivity: >0.72). Exogenous miR-146a expression is significantly increased in vitro oncogenic phenotypes as well as during xenograft tumorigenesis and OSCC metastasis. The plasma miR-146a levels of these mice parallel the xenograft tumor burdens of the mice. A miR-146a blocker abrogates the growth of xenograft tumors. miR-146a oncogenic activity is associated with down-regulation of IRAK1, TRAF6 and NUMB expression. Furthermore, miR-146a directly targets the 3′UTR of NUMB and a region within the NUMB coding sequence when suppressing NUMB expression. Exogenous NUMB expression attenuates OSCC oncogenicity. Double knockdown of IRAK1 and TRAF6, and of TRAF6 and NUMB, enhance the oncogenic phenotypes of OSCC cells. Oncogenic enhancement modulated by miR-146a expression is attenuated by exogenous IRAK1 or NUMB expression. This study shows that miR-146a expression contributes to oral carcinogenesis by targeting the IRAK1, TRAF6 and NUMB genes.

Smurf2-mediated degradation of EZH2 enhances neuron differentiation and improves functional recovery after ischaemic stroke.

EZH2 plays an important role in stem cell renewal and maintenance by inducing gene silencing via its histone methyltransferase activity. Previously, we showed that EZH2 downregulation enhances neuron differentiation of human mesenchymal stem cells (hMSCs); however, the underlying mechanisms of EZH2‐regulated neuron differentiation are still unclear. Here, we identify Smurf2 as the E3 ubiquitin ligase responsible for the polyubiquitination and proteasome‐mediated degradation of EZH2, which is required for neuron differentiation. A ChIP‐on‐chip screen combined with gene microarray analysis revealed that PPARγ was the only gene involved in neuron differentiation with significant changes in both its modification and expression status during differentiation. Moreover, knocking down PPARγ prevented cells from undergoing efficient neuron differentiation. In animal model, rats implanted with intracerebral EZH2‐knocked‐down hMSCs or hMSCs plus treatment with PPARγ agonist (rosiglitazone) showed better improvement than those without EZH2 knockdown or rosiglitazone treatment after a stroke. Together, our results support Smurf2 as a regulator of EZH2 turnover to facilitate PPARγ expression, which is specifically required for neuron differentiation, providing a molecular mechanism for clinical applications in the neurodegenerative diseases.

miR-211 promotes the progression of head and neck carcinomas by targeting TGFβRII

miR-211 up-regulation and transforming growth factor-β type II receptor (TGFβRII) down-regulation are associated with poor prognosis of head and neck squamous cell carcinoma (HNSCC). miR-211 directly targets TGFβRII with the miR-211-TGFβRII-c-Myc axis promoting HNSCC progression. An inverse correlation of miR-211 and TGFβRII expression was found in metastatic HNSCC samples. After 4-nitroquinoline 1-oxide induction, more severe epithelial tumorigenesis was detected on K14-miR-211 transgenic mouse dorsal tongues. Human metastatic lesions and mouse tongue tumors showed increased nuclear c-Myc expression. A novel role for miR-211 in the regulation of TGFβRII and c-Myc during tumorigenesis being revealed should help to develop anti-HNSCC therapies.

The Association between Genetic Polymorphism and the Processing Efficiency of miR-149 Affects the Prognosis of Patients with Head and Neck Squamous Cell Carcinoma

MicroRNAs (miRNAs) play important roles in modulating the neoplastic process of cancers including head and neck squamous cell carcinoma (HNSCC). A genetic polymorphism (rs2292832, C>T) has been recently identified in the precursor of miR-149; nevertheless its clinicopathological implications remain obscure. In this study, we showed that miR-149 is down-regulated in HNSCC compared to normal mucosa and this is associated with a poorer patient survival. In addition, HNSCC patients with the T/T genotype have more advanced tumors and a worse prognosis. Multivariate analysis indicated that patients carried the T/T genotype have a 2.81-fold (95% CI: 1.58–4.97) increased risk of nodal metastasis and 1.66-fold (95% CI: 1.05–2.60) increased risk of mortality compared to other groups. T/T genotype also predicted the worse prognosis of buccal mucosa carcinoma subset of HNSCC. In vitro analysis indicated that exogenous miR-149 expression reduces the migration of HNSCC cells. Moreover, HNSCC cell subclones carrying the pri-mir-149 sequence containing the T variant show a low processing efficacy when converting the pre-mir-149 to mature miR-149. These findings suggest that miR-149 suppresses tumor cell mobility, and that the pre-mir-149 polymorphism may affect the processing of miR-149, resulting in a change in the abundance of the mature form miRNA, which, in turn, modulates tumor progression and patient survival.

Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

Receptor tyrosine kinases (RTKs) are cell surface receptors that initiate signal cascades in response to ligand stimulation. Abnormal expression and dysregulated intracellular trafficking of RTKs have been shown to be involved in tumorigenesis. Recent evidence shows that these cell surface receptors translocate from cell surface to different cellular compartments, including the Golgi, mitochondria, endoplasmic reticulum (ER) and the nucleus, to regulate physiological and pathological functions. Although some trafficking mechanisms have been resolved, the mechanism of intracellular trafficking from cell surface to the Golgi is not yet completely understood. Here we report a mechanism of Golgi translocation of epidermal growth factor receptor (EGFR) in which EGF-induced EGFR travels to the Golgi via microtubule-dependent movement by interacting with dynein and fuses with the Golgi through syntaxin 6-mediated membrane fusion. We also demonstrate that the microtubule- and syntaxin 6-mediated Golgi translocation of EGFR is necessary for its consequent nuclear translocation and nuclear functions. Thus, together with previous studies, the microtubule- and syntaxin 6-mediated trafficking pathway from cell surface to the Golgi, ER and the nucleus defines a comprehensive trafficking route for EGFR to travel from cell surface to the Golgi and the nucleus.

Distinct Subpopulations of Head and Neck Cancer Cells with Different Levels of Intracellular Reactive Oxygen Species Exhibit Diverse Stemness, Proliferation, and Chemosensitivity

Head and neck squamous cell carcinoma (HNSCC) is driven by cancer-initiating cells (CIC), but their maintenance mechanisms are obscure. For hematopoietic stem cells, low levels of intracellular reactive oxygen species (ROS(Low)) is known to help sustain stemness properties. In this report, we evaluated the hypothesis that ROS(Low) character conferred CIC properties in HNSCC. Sphere cultures define CIC in HNSCC cell populations (HN-CIC). We found that ROS(Low) cells in HN-CIC defined in this manner were more numerous than in parental HNSCC cells. Further, ROS(Low) cells frequently coexpressed CIC surface markers such as memGrp78 and Glut3. Exploiting flow cytometry to sort cells on the basis of their ROS level, we found that isolated ROS(Low) cells displayed relatively more CIC properties, including quiescence, chemoresistance, in vitro malignant properties, and tumorigenicity. Pharmacological depletion of ROS modulators in cisplatin-treated HN-CIC reduced CIC properties, enhancing cell differentiation and enhancing cisplatin-induced cell death. Overall, our work defined cell subpopulations in HNSCC on the basis of differential intracellular ROS levels, which associated with stemness and chemoresistance properties. On the basis of our findings, we suggest that strategies to promote intracellular ROS levels may heighten the efficacy of conventional chemotherapy used for HNSCC treatment.