Bin Qiao

Cancer stem cell: Fundamental experimental pathological concepts and updates

Cancer stem cells (CSCs) are a subset of cancer cells which play a key role in predicting the biological aggressiveness of cancer due to its ability of self-renewal and multi-lineage differentiation (stemness). The CSC model is a dynamic one with a functional subpopulation of cancer cells rather than a stable cell population responsible for tumour regeneration. Hypotheses regarding the origins of CSCs include (1) malignant transformation of normal stem cells; (2) mature cancer cell de-differentiation with epithelial-mesenchymal transition and (3) induced pluripotent cancer cells. Surprisingly, the cancer stem cell hypothesis originated in the late nineteenth century and the existence of haematopoietic stem cells was demonstrated a century later, demonstrating that the concept was possible. In the last decade, CSCs have been identified and isolated in different cancers. The hallmark traits of CSCs include their heterogeneity, interaction with microenvironments and plasticity. Understanding these basic concepts of CSCs is important for translational applications using CSCs in the management of patients with cancer.

Three-dimensional CT angiography for the diagnosis and assessment of arteriovenous malformations in the oral and maxillofacial region

OBJECTIVE The purpose of this study was to evaluate the diagnostic performance of three-dimensional computed tomography angiography (3D-CTA) for arteriovenous malformations (AVMs) in the oral and maxillofacial region. MATERIALS AND METHODS Sixty four-slice spiral CT angiography of oral or maxillofacial region was performed in 8 patients with surgically proven arteriovenous malformations. The morphologic features, size, location, boundary, and feeding and draining vessels of lesions were reviewed. RESULTS AVMs in 5 patients were located in the soft tissues and 3 were in the mandible. CTA of all cases showed tangles of disorganized vessels with well-defined borders. The feeding and draining vessels were enlarged and tortuous. Four patients had bone involvement. CONCLUSION Sixty four-slice spiral CTA can accurately demonstrate the morphological characteristics of AVMs.

BMI-1 activation is crucial in hTERT-induced epithelial-mesenchymal transition of oral epithelial cells.

BMI-1 (B lymphoma Mo-MLV insertion region 1 homolog) has been reported to be over-expressed in cell immortalisation and the epithelial-mesenchymal transition (EMT) of cancer cells. The aim of this study is to study the roles of BMI-1 in the human telomerase reverse transcriptase (hTERT)-induced immortalisation and EMT. In this study, hTERT(+)-OME cells and hTERT(+)-HaCaT cells were acquired by viral transduction of hTERT to primary cultured oral keratinocytes and HaCaT cells (skin epidermal cells). siRNA transduction was used for the inhibition of BMI-1 expression. RT-PCR and Western blots were performed to detect the expressions of twist, vimentin, BMI-1, hTERT and p16INK4a in these cell lines. EMT was assessed by immunohistochemistry (expressions of cytokertin & vimentin), Western blots (expressions of Twist, vimentin & E-cadherin) and RT-PCR (expression of Twist). The results indicated that hTERT(+)-OME cells and hTERT(+)-HaCaT cells underwent EMT spontaneously with high expression of Twist. p16INK4a was silenced in both hTERT-transduced cells but could be detected in HaCaT cells. Moreover, BMI-1 was highly expressed in hTERT(+)-OME and hTERT(+)-HaCaT cells but was negative in HaCaT cells. When the expression of BMI-1 was blocked by siRNA transduction, the proliferations of hTERT(+)-OME and hTERT(+)-HaCaT cells were inhibited and the mono-spheroid colony formation of these hTERT-transduced cells was decreased. In addition, the expression of p16INK4a was regained while the expressions of EMT markers (twist and vimentin) were down-regulated in these two BMI-1 blocking cell lines. To conclude, this study suggests BMI-1 expression plays a role in hTERT-induced immortalisation and EMT.

Epithelial-mesenchymal transition and mesenchymal-epithelial transition are essential for the acquisition of stem cell properties in hTERT-immortalised oral epithelial cells.

Evidence has shown that mesenchymal–epithelial transition (MET) and epithelial–mesenchymal transition (EMT) are linked to stem cell properties. We currently lack a model showing how the occurrence of MET and EMT in immortalised cells influences the maintenance of stem cell properties. Thus, we established a project aiming to investigate the roles of EMT and MET in the acquisition of stem cell properties in immortalised oral epithelial cells.

Stage dependent expression and tumor suppressive function of FAM134B (JK1) in colon cancer.

The aims of the present study are to investigate sub‐cellular location, differential expression in different cancer stages and functional role of FAM134B in colon cancer development. FAM134B expression was studied and quantified at protein and mRNA levels in cell lines using immunocytochemistry, Western blot and real‐time PCR. In vitro functional assays and an in vivo xenotransplantation mouse models were used to investigate the molecular role of FAM134B in cancer cell biology in response to FAM134B silencing with shRNA lentiviral particles. FAM134B protein was noted in both cytoplasm and nuclei of cancer cells. In cancer cells derived from stage IV colon cancer, FAM134B expression was remarkably reduced when compared to non‐cancer colon cells and cancer cells derived from stage II colon cancer. FAM134B knockdown significantly (P < 0.05) increased the proliferation of colon cancer cells following lentiviral transfection. Furthermore, FAM134B suppression significantly increased (34–52%; P < 0.05) the clonogenic capacity, wound healing potential of and increases the proportion of cells performing DNA synthesis (P < 0.01). Xenotransplantation model showed that larger and higher‐grade tumors were formed in mice receiving FAM134B knockdown cells. To conclude, expression analysis, in vitro and in vivo indicated that FAM134B acts as a cancer suppressor gene in colon cancer.

p63 and its isoforms as markers of rat oral mucosa epidermal stem cells in vitro.

Although techniques for purifying epidermal stem cells (ESCs) have been established, enriching a pure population of viable ESCs is still a challenging task. One approach toward enhancing the purity and viability of ESCs involves cell markers. While evidence suggests that p63 plays a role in maintaining the population of ESCs, whether p63 can function as a specific marker for ESCs is unclear. We isolated and cultured oral ESCs and illustrated the expression of p63 and its isoforms in rat oral mucosa tissues and stem cells before and after differentiation. Semi‐reverse transcription PCR detected the TA, ΔN, α and β isoforms when cells were cultured for 2 days, but only TA and γ were detected after 14 days. We also found that p63 is expressed in basal and suprabasal epithelial layers of rat oral mucosa, but it was implied p63 does not mark stem cells specifically, while the ΔNp63α and ΔNp63β isoforms may be specific markers of rat oral mucosa stem cells. Copyright

Telomerase reverse transcriptase potentially promotes the progression of oral squamous cell carcinoma through induction of epithelial-mesenchymal transition

In recent years, researchers have found the critical role of telomerase in cellular transformation, proliferation, stemness and cell survival. High levels of telomerase reverse transcriptase (TERT) expression and telomerase activation have been reported in most cancer cells. Moreover, overexpression of human TERT (hTERT) is reported to be correlated with advanced invasive stage of the tumor progression and poor prognosis. Epithelial-mesenchymal transition (EMT), characterized by the loss of the cell-cell contact of epithelial cells and the acquisition of migratory and motile properties, is known to be a central mechanism responsible for invasiveness and metastasis of various cancers. Thus, we investigated whether hTERT plays a potential role in the development of EMT. As we expected, our clinical results showed that hTERT is overexpressed in oral epithelial dysplasia (OED) and OSCC tissues and correlates with clinical aggressiveness of oral squamous cell carcinoma (OSCC) patients. We then overexpressed hTERT in primary human oral epithelial cells (HOECS) and found that hTERT has the potential to prolong the lifespan, a process confering the characteristics of EMT by activating the Wnt/β-catenin pathway. Our findings provided an explanation for the aggressive nature of human tumors overexpressing hTERT and the possibly mechanism that links hTERT to EMT property, which represents a possible therapeutic target in highly metastatic cancers.

MicroRNA-27a-3p Modulates the Wnt/β-Catenin Signaling Pathway to Promote Epithelial-Mesenchymal Transition in Oral Squamous Carcinoma Stem Cells by Targeting SFRP1.

This study aimed to elucidate how microRNA27a-3p (miR-27a-3p) modulates the Wnt/β-catenin signaling pathway to promote the epithelial-mesenchymal transition (EMT) in oral squamous carcinoma stem cells (OSCSCs) by targeting secreted frizzled-related protein 1 (SFRP1). Flow cytometry was used to sort OSCSCs from the SCC-9 and Tca8113 cell lines. The OSCSCs were randomly assigned into the miR-27a-3p inhibitors group, the miR-27a-3p inhibitors-NC group, the si-SFRP1 group, the si-SFRP1 + miR-27a-3p inhibitors group and the blank group. A luciferase reporter, immunofluorescence and Transwell assays were performed to detect luciferase activity, SFRP1, and cell migration and invasion, respectively. The mRNA expression of miR-27a-3p, SFRP1 and EMT markers (E-cadherin, N-cadherin, vimentin and ZEB1) were detected using qRT-PCR. The protein expression of SFRP1, EMT markers and the proteins of the Wnt/β-catenin signaling pathway was detected by Western blotting. OSCSCs showed up-regulated miR-27a-3p, Wnt/β-catenin signaling pathway-related proteins, vimentin, N-cadherin and ZEB1 and down-regulated SFRP1 and E-cadherin. MiR-27a-3p targeted SFRP1. Down-regulated miR-27a-3p resulted in increased E-cadherin and SFRP1 but decreased vimentin, N-cadherin, ZEB1, the Wnt/β-catenin signaling pathway-related proteins, and invasive and migratory cells. Silenced SFRP1 reversed this effect. We found that miR-27a-3p modulated the Wnt/β-catenin signaling pathway to promote EMT in OSCSCs by down-regulating SFRP1.

Regulation of tumorigenesis in oral epithelial cells by defined reprogramming factors Oct4 and Sox2

Oct4 and Sox2 are pluripotent stem cell factors but the interplay between them in tumorigenesis is unclear. The aim of the present study was to investigate the roles of Oct4 and Sox2 in the reprogramming of oral cancer stem cells. One or both Oct4 and Sox2 were overexpressed in immortalized oral epithelial (hTERT+-OME) cells by lentivirus transduction. In addition, Oct4 and Sox2 proteins in two oral squamous cell carcinoma cell (OSCC) lines (Cal27 and primary cultured OSCC from a T2N2M0 patient) were individually or combinedly knocked down by shRNA. The results showed that the doubly transduced (Oct4+Sox2+) cells could trigger neoplasms in immunodeficient mice after lentivirus transduction, but single transduced (Oct4+ or Sox2+) cells had no tumor formation ability. The knockdown Sox2low and knockdown Oct4lowSox2low cells resulted in decreased tumor size in the immunodeficient mice but the single knockdown Oct4low cancer cells acquired more aggressive xenografts. Our findings suggest that Oct4+Sox2+ cells may be reprogrammed cancer stem cells inducing oral carcinogenesis.

Sox2 promotes tumor aggressiveness and epithelial‑mesenchymal transition in tongue squamous cell carcinoma

Tongue squamous cell carcinoma (TSCC) is highly malignant and poorly differentiated, resulting in a high frequency of local recurrence and distant metastases. Sox2 (Sry-box2), an important factor in embryonic development and cell differentiation, has been shown to associate with malignant phenotypes and epithelial-mesenchymal transition (EMT) progression in numerous types of human tumors. However, the clinical relevance and molecular mechanisms of Sox2 in TSCC remain unclear. In the present study, the expression levels of Sox2 were assessed in 61 pairs of TSCC samples and corresponding adjacent non-cancerous tissues using immunohistochemical methods. Associations between Sox2 expression and clinicopathological features were evaluated. Furthermore, Sox2 was overexpressed and inhibited using full-length Sox2 cDNA and short hairpin RNA (shRNA) transfection in UM2 and Cal27 cell lines, respectively. The malignant phenotypes were assessed by plate clone formation assays, wound-healing assays and Transwell assays. EMT markers (E-cadherin, vimentin, Twist, Slug and Snail) and β-catenin were detected by reverse transcription-polymerase chain reaction and western blot analysis following the alterations of Sox2 expression. The results indicated that Sox2 expression was markedly upregulated in TSCC samples and was significantly associated with tumor growth (pT stage), cell differentiation, lymphatic metastasis (pN stage) and clinical stage (pTNM stage). Cal27-shRNA-Sox2 cells not only exhibited a decreased capacity for cell proliferation, but also suppressed cell migration and invasion, and an attenuated colony formation capacity. By contrast, UM2-Sox2 cells exhibited accelerated cell malignant phenotypes and EMT progression. Moreover, when the expression of Sox2 was decreased by shRNA transduction, β-catenin expression was attenuated. An opposing phenomenon was observed in UM2-Sox2 cells. In conclusion, this study suggests that Sox2 expression serves a role in TSCC malignant phenotypes and EMT progression, and that β-catenin may act as a modulated factor in this progression.