Ya-ling Tang

EMT: a new vision of hypoxia promoting cancer progression.

A hypoxic microenvironment plays a critical role in the development and progression of tumors. The epithelial to mesenchymal transition (EMT) is a process by which epithelial cells lose their polarity and are converted to a mesenchymal phenotype, which is regarded as a critical event in morphogenetic changes during embryonic development, wound healing, and cancer metastasis. Recent advances in our understanding of the molecular pathways that govern the association of hypoxia with malignant tumors point to the epithelial to mesenchymal transition (EMT). The hypoxic microenvironment common to cancer cells emerges as an important factor in the induction of a pathological EMT, which is a key link in cancer progression. This review presents the potential molecular mechanisms underlying the hypoxia/HIF-dependent regulation of the EMT in cancer.

Hypoxia-inducible factor-1 alpha, in association with TWIST2 and SNIP1, is a critical prognostic factor in patients with tongue squamous cell carcinoma

It has become apparent that hypoxia and hypoxia-inducible factor-1 (HIF-1) activation have the potential of modulating the activity of major epithelial-mesenchymal transition (EMT)-triggering pathways by regulating the expression and activity levels of major transcriptional repressors. The aim of our study was to elucidate the role of HIF-1α and HIF-2α, and EMT regulators TWIST2 and SMAD nuclear interacting protein-1 (SNIP1) in tongue squamous cell carcinoma (TSCC). A retrospective analysis of 89 patients with TSCC from Department of Oral and Maxillofacial Surgery, West China Hospital of Stomatology, Sichuan University between 2002 and 2005 was performed using immunohistochemistry in paraffin-embedded and formalin-fixed tissues to analyze HIF-1α, HIF-2α, TWIST2 and SNIP1 expression. The association between HIF-1α, HIF-2α, TWIST2 and SNIP1 expression and patient survivals was investigated. Our results showed that overexpression of HIF-1α, HIF-2α, TWIST2 and SNIP1 were shown in 49.44% (44/89), 55.06% (49/89), 44.94% (40/89) and 34.83% (31/89) of TSCC, respectively. Overexpression of HIF-1α, TWIST2 and SNIP1 in TSCC was associated with a shorter disease-free survival (P=0.003, P=0.001, P=0.040, respectively), and HIF-2α had no significant association with either overall survival (P=0.195) or disease-free survival (P=0.356). Co-expression of more than two markers of HIF-1α, TWIST2 and SNIP1 was an independent prognostic indicator for both overall survival and disease-free survival by multivariate Cox proportional hazards model. It is proposed that co-expression of more than two markers from HIF-1α, TWIST2 and SNIP1 might be a significant prognostic predictor in patients with TSCC.

Inflammation linking EMT and cancer stem cells

Similar to actors changing costumes during a performance, cancer cells undergo many rapid changes during the process of tumor metastasis, including epithelial-mesenchymal transition (EMT), acquisition of cancer stem cells (CSCs) properties, and mesenchymal-epithelial transition (MET). Such changes allow the tumor to compete with the normal microenvironment to overcome anti-tumorigenic pressures. Then, once tissue homeostasis is lost, the altered microenvironment, like that accompanying inflammation, can itself become a potent tumor promoter. This review will discuss the changes that cancer cells undergo in converting from EMT to CSCs in an inflammation microenvironment, to understand the mechanisms behind invasion and metastasis and provide insights into prevention of metastasis.

microRNAs, an active and versatile group in cancers

microRNAs (miRNAs) are a class of non‐coding RNAs that function as endogenous triggers of the RNA interference pathway. Studies have shown that thousands of human protein‐coding genes are regulated by miRNAs, indicating that miRNAs are master regulators of many important biological processes, such as cancer development. miRNAs frequently have deregulated expression in many types of human cancers, and play critical roles in tumorigenesis, which functions either as tumor suppressors or as oncogenes. Recent studies have shown that miRNAs are highly related with cancer progression, including initiating, growth, apoptosis, invasion, and metastasis. Furthermore, miRNAs are shown to be responsible for the cancer‐related inflammation, anti‐cancer drug resistance, and regulation of cancer stem cells. Therefore, miRNAs have generated great interest as a novel strategy in cancer diagnosis and therapy. Here we review the versatile roles of miRNAs in cancers and their potential applications for diagnosis, prognosis, and treatment as biomarkers.

Expression of c-kit and Slug correlates with invasion and metastasis of salivary adenoid cystic carcinoma

The overexpression of c-kit seems to be frequent and specific in salivary adenoid cystic carcinoma (ACC), however, there is little information on correlation between c-kit expression and the invasion and metastasis. Recently, the data showed that Slug, a transcription factor of epithelial-mesenchymal transitions (EMT), is a molecular target that contributes to the biological specificity of c-kit signaling pathway. In this study, the expression of c-kit and Slug was evaluated in two ACC cell lines and 121 patients with ACC. The results of real-time RT-PCR and Western blot showed that ACC-2 and ACC-M cell lines expressed c-kit and Slug mRNA and protein. The immunohistochemical assay in patients demonstrated that positive expression of c-kit and Slug was observed in 108/121 (89.26%) and 87/121 (71.90%) of cases, respectively, and that c-kit and Slug expression was significantly associated with tumor site, TNM stage, histological pattern, perineural invasion, local regional recurrence and distant metastasis of patients with ACC (P<0.05). Furthermore, there was a significant association between the positive expression of c-kit and that of Slug (P=0.046). These findings indicated that c-kit/Slug pathway might participate in the invasion and metastasis of salivary ACC.

Hypoxia Inducible Factor 1α and Hypoxia Inducible Factor 2α Play Distinct and Functionally Overlapping Roles in Oral Squamous Cell Carcinoma

Purpose: This study aimed to investigate the functional difference between hypoxia inducible factor (HIF)-1α and HIF-2α in oral squamous cell carcinomas (OSCC). Experimental Design: We evaluated the correlations between HIF-1α and HIF-2α expression and the clinical-pathologic characteristics of 97 patients with OSCC by immunohistochemical staining. OSCC cell lines transfected with lentivirus encoding short hairpin RNA against HIF-1α/2α were used to investigate the HIF-1α/2α–dependent target genes. Xenograft tumors in nude mice were established using cells affected by lentivirus, and tumor growth, angiogenesis, proliferation, and apoptosis were measured. Results: HIF-1α expression was significantly associated with T stage (P = 0.004), lymph node involvement (P = 0.006), histologic differentiation (P = 0.013), and microvessel density (P = 0.014), whereas that of HIF-2α was associated with T stage (P = 0.011) and microvessel density (P = 0.005). Patients with positive HIF-1α nuclear staining had a significantly worse overall survival (P < 0.001) and disease-free survival (P < 0.001) than those with negative HIF-1α staining. When OSCC cells were cultured at 5% O2, only HIF-2α contributed to the expression of vascular endothelial growth factor. At 1% O2, vascular endothelial growth factor was regulated by both HIF-1α and HIF-2α, but glucose transporter 1, carbonic anhydrase 9, and urokinase-type plasminogen activator receptor were regulated by HIF-1α rather than by HIF-2α. Knocking down HIF-1α or HIF-2α individually inhibited the xenograft tumor angiogenesis and growth, and knocking them down simultaneously revealed a better inhibitory effect than knocking down either unit alone. Conclusions: HIF-1α and HIF-2α correlated with different clinical-pathologic parameters, stabilized at different oxygen levels, and regulated different genes in OSCC. However, both HIF-1α and HIF-2α showed promoting roles in tumor angiogenesis and growth, and therapeutic outcome may benefit from combined targeting of HIF-1α and HIF-2α. Clin Cancer Res; 16(19); 4732–41. ©2010 AACR.

RNAi targeting urokinase-type plasminogen activator receptor inhibits metastasis and progression of oral squamous cell carcinoma in vivo

It has been admitted that urokinase‐type plasminogen activator receptor (u‐PAR) is overexpressed in many human malignant tumors including oral squamous cell carcinoma (OSCC) and plays an important role in a variety of cancer key cellular events as a versatile signaling orchestrator. In our study, a retroviral vector expressing u‐PAR‐specific siRNA was injected into OSCC xenografts of nude mice to observe its inhibitory effects on OSCC. Our data demonstrate that siRNA targeting u‐PAR markedly suppressed tumor growth, reduced the expression of proliferation‐related gene, Ki‐67 and increased cell apoptosis, accompanying with the efficient and specific inhibition of endogenous u‐PAR expression in OSCC. More importantly, the mRNA and protein expression of MMP‐2, MMP‐9, VEGF‐C, VEGF‐D and VEGFR‐3 that are intimately involved in oral cancer invasion and metastasis, was simultaneously downregulated significantly as determined by quantitative real‐time RT‐PCR, Western blot and immunohistochemistry; and Gelatin and fibrin zymography showed that MMP‐9, MMP‐2 and u‐PA enzymatic activities were significantly reduced in u‐PAR‐specific siRNA group, compared to those in control groups. In addition, the expression of MDR‐1 gene related to drug resistance was obviously inhibited by silencing of u‐PAR. These findings suggest that RNAi targeting u‐PAR could effectively inhibit the metastasis and progression of OSCC in vivo. Thus, it may be used as a potent and specific therapy for oral cancer, especially in inhibiting and preventing cancer cell invasion and metastasis.

RNAi-mediated downregulation of urokinase plasminogen activator receptor inhibits proliferation, adhesion, migration and invasion in oral cancer cells.

RNA interference (RNAi) has emerged as an effective method to target specific genes for silencing. Overexpression of urokinase-type plasminogen activator receptor (uPAR) has been implicated in progression and metastasis of oral cancer. In our study, RNAi was introduced to downregulate the expression of uPAR in the highly malignant oral squamous cell carcinoma (OSCC) cells. Our data demonstrated that siRNA targeting of uPAR leads to the efficient and specific inhibition of endogenous uPAR mRNA and protein expression as determined by quantitative real-time RT-PCR and Western blotting. Furthermore, simultaneous silencing of uPAR resulted in a dramatic reduction of tumor cell proliferation activity, adhesion, migration and invasion in vitro compared to the controls. These findings provide further evidence for the involvement of uPAR in a variety of cancer key cellular events as a versatile signaling orchestrator, and suggest that RNAi-directed targeting of uPAR can be used as a potent and specific therapeutic tool for the treatment of oral cancer, especially in inhibiting and/or preventing cancer cell invasion and metastasis.

HIF-α/MIF and NF-κB/IL-6 Axes Contribute to the Recruitment of CD11b+Gr-1+ Myeloid Cells in Hypoxic Microenvironment of HNSCC

CD11b+Gr-1+ myeloid cells have gained much attention due to their roles in tumor immunity suppression as well as promotion of angiogenesis, invasion, and metastases. However, the mechanisms by which CD11b+Gr-1+ myeloid cells recruit to the tumor site have not been well clarified. In the present study, we showed that hypoxia could stimulate the migration of CD11b+Gr-1+ myeloid cells through increased production of macrophage migration inhibitory factor (MIF) and interleukin-6 (IL-6) by head and neck squamous cell carcinoma (HNSCC) cells. Hypoxia-inducible factor-1α (HIF-1α)- and HIF-2α-dependent MIF regulated chemotaxis, differentiation, and pro-angiogenic function of CD11b+Gr-1+ myeloid cells through binding to CD74/CXCR2, and CD74/CXCR4 complexes, and then activating p38/mitogen-activated protein kinase (MAPK) and phosphatidylinositide 3-kinases (PI3K)/AKT signaling pathways. Knockdown (KD) of HIF-1α and HIF-2α in HNSCC cells decreased MIF level but failed to inhibit the CD11b+Gr-1+ myeloid cell migration, because HIF-1α/2α KD enhanced nuclear factor κB (NF-κB) activity that increased IL-6 secretion. Simultaneously blocking NF-κB and HIF-1α/HIF-2α had better inhibitory effect on CD11b+Gr-1+ myeloid cell recruitment in the hypoxic zone than individually silencing HIF-1α/2α or NF-κB. In conclusion, the interaction between HIF-α/MIF and NF-κB/IL-6 axes plays an important role in the hypoxia-induced accumulation of CD11b+Gr-1+ myeloid cells and tumor growth in HNSCC.

The crosstalk between lncRNA and microRNA in cancer metastasis: orchestrating the epithelial-mesenchymal plasticity

Noncoding RNAs (ncRNAs) have been demonstrated to closely associate with gene regulation and encompass the well-known microRNAs (miRNAs), as well as the most recently acknowledged long noncoding RNAs (lncRNAs). Current evidence indicates that lncRNAs can interact with miRNAs and these interactions play crucial roles in cancer metastasis, through regulating critical events especially the epithelial-mesenchymal transition (EMT). This review summarizes the types of lncRNA-miRNA crosstalk identified to-date and discusses their influence on the epithelial-mesenchymal plasticity and clinical metastatic implication.