Cheng-Chia Yu

PURPOSE: Oral squamous cell carcinoma (OSCC), like many solid tumors, contains a heterogeneous population of cancer cells. Recent data suggest that a rare subpopulation of cancer cells, termed cancer stem cells (CSC), is capable of initiating, maintaining, and expanding the growth of tumor. Identification and characterization of CSC from OSCC facilitates the monitoring, therapy, or prevention of OSCC. EXPERIMENTAL DESIGN: We enriched oral cancer stem-like cells (OC-SLC) through sphere formation by cultivating OSCC cells from established OSCC cell lines or primary cultures of OSCC patients within defined serum-free medium. Differential expression profile of stemness genes between enriched OC-SLC and parental OSCC was elucidated. Furthermore, immunohistochemical staining of stemness markers on OSCC patient tissues was examined to evaluate the association between stemness genes and prognosis of OSCC. RESULTS: Enriched OC-SLC highly expressed the stem/progenitor cell markers and ABC transporter gene (Oct-4, Nanog, CD117, Nestin, CD133, and ABCG2) and also displayed induced differentiation abilities and enhanced migration/invasion/malignancy capabilities in vitro and in vivo. Elevated expression of CD133 was shown in the enriched OC-SLC from OSCC patients' tumors. Positive correlations of Oct-4, Nanog, or CD133 expression on tumor stage were shown on 52 OSCC patient tissues. Kaplan-Meier analyses exhibited that Nanog/Oct-4/CD133 triple-positive patients predicted the worst survival prognosis of OSCC patients. CONCLUSION: We enriched a subpopulation of cancer stem-like cell from OSCC by sphere formation. The enriched OC-SLC possesses the characteristics of both stem cells and malignant tumors. Additionally, expression of stemness markers (Nanog/Oct-4/CD133) contradicts the survival prognosis of OSCC patients.

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 OSCC's recurrence to resist cisplatin.

Cancer-initiating cells (CIC) comprise a rare subpopulation of cells in tumors that are proposed to be responsible for tumor growth. Starting from CICs identified in head and neck squamous cell carcinomas (HNSCC), termed head and neck cancer-initiating cells (HN-CIC), we determined as a candidate stemness-maintaining molecule for HN-CICs the proinflammatory mediator S100A4, which is also known to be an inducer of epithelial-mesenchymal transition. S100A4 knockdown in HN-CICs reduced their self-renewal capability and their stemness and tumorigenic properties, both in vitro and in vivo. Conversely, S100A4 overexpression in HNSCC cells enhanced their stem cell properties. Mechanistic investigations indicated that attenuation of endogenous S100A4 levels in HNSCC cells caused downregulation of Notch2 and PI3K (phosphoinositide 3-kinase)/pAKT along with upregulation of PTEN, consistent with biological findings. Immunohistochemical analysis of HNSCC clinical specimens showed that S100A4 expression was positively correlated with clinical grading, stemness markers, and poorer patient survival. Together, our findings reveal a crucial role for S100A4 signaling pathways in maintaining the stemness properties and tumorigenicity of HN-CICs. Furthermore, our findings suggest that targeting S100A4 signaling may offer a new targeted strategy for HNSCC treatment by eliminating HN-CICs.

Lin28, a key factor for cellular reprogramming and generation of induced pluripotent stem cell (iPSC), makes a critical contribution to tumorigenicity by suppressing Let-7. However, it is unclear whether Lin28 is involved in regulating cancer stem-like cells (CSC), including in oral squamous carcinoma cells (OSCC). In this study, we demonstrate a correlation between high levels of Lin28B, Oct4, and Sox2, and a high percentage of CD44(+)ALDH1(+) CSC in OSCC. Ectopic Lin28B expression in CD44(-)ALDH1(-)/OSCC cells was sufficient to enhance Oct4/Sox2 expression and CSC properties, whereas Let7 co-overexpression effectively reversed these phenomena. We identified ARID3B and HMGA2 as downstream effectors of Lin28B/Let7 signaling in regulating endogenous Oct4 and Sox2 expression. Let7 targeted the 3' untranslated region of ARID3B and HMGA2 and suppressed their expression, whereas ARID3B and HMGA2 increased the transcription of Oct4 and Sox2, respectively, through promoter binding. Chromatin immunoprecipitation assays revealed a direct association between ARID3B and a specific ARID3B-binding sequence in the Oct4 promoter. Notably, by modulating Oct4/Sox2 expression, the Lin28B-Let7 pathway not only regulated stemness properties in OSCC but also determined the efficiency by which normal human oral keratinocytes could be reprogrammed to iPSC. Clinically, a Lin28B(high)-Let7(low) expression pattern was highly correlated with high levels of ARID3B, HMGA2, OCT4, and SOX2 expression in OSCC specimens. Taken together, our results show how Lin28B/Let7 regulates key cancer stem-like properties in oral squamous cancers.