Molecular signaling in cancer stem cells of tongue squamous cell carcinoma: Therapeutic implications and challenges

Head and neck squamous cell carcinoma is the seventh most common cancer worldwide with high mortality rates. Amongst oral cavity cancers, tongue carcinoma is a very common and aggressive oral cavity carcinoma. Despite the implementation of a multimodality treatment regime including surgical intervention, chemo-radiation as well as targeted therapy, tongue carcinoma shows a poor overall 5-year survival pattern, which is attributed to therapy resistance and recurrence of the disease. The presence of a rare population, i.e., cancer stem cells (CSCs) within the tumor, are involved in therapy resistance, recurrence, and distant metastasis that results in poor survival patterns. Therapeutic agents targeting CSCs have been in clinical trials, although they are unable to reach into therapy stage which is due to their failure in trials. A more detailed understanding of the CSCs is essential for identifying efficient targets. Molecular signaling pathways, which are differentially regulated in the CSCs, are one of the promising targets to manipulate the CSCs that would provide an improved outcome. In this review, we summarize the current understanding of molecular signaling associated with the maintenance and regulation of CSCs in tongue squamous cell carcinoma in order to emphasize the need of the hour to get a deeper understanding to unravel novel targets.     

Up-regulation of TNF-alpha/NFkB/SIRT1 axis drives aggressiveness and cancer stem cells accumulation in chemoresistant oral squamous cell carcinoma

Tumor resistance remains an obstacle to successfully treating oral squamous cell carcinoma (OSCC). Cisplatin is widely used as a cytotoxic drug to treat solid tumors, including advanced OSCC, but with low efficacy due to chemoresistance. Therefore, identifying the pathways that contribute to chemoresistance may show new possibilities for improving the treatment. This work explored the role of the tumor necrosis factor-alpha (TNF-alpha)/NFkB signaling in driving the cisplatin resistance of OSCC and its potential as a pharmacological target to overcome chemoresistance. Differential accessibility analysis demonstrated the enrichment of opened chromatin regions in members of the TNF-alpha/NFkB signaling pathway, and RNA-Seq confirmed the upregulation of TNF-alpha/NFkB signaling in cisplatin-resistant cell lines. NFkB was accumulated in cisplatin-resistant cell lines and in cancer stem cells (CSC), and the administration of TNF-alpha increased the CSC, suggesting that TNF-alpha/NFkB signaling is involved in the accumulation of CSC. TNF-alpha stimulation also increased the histone deacetylases HDAC1 and SIRT1. Cisplatin-resistant cell lines were sensitive to the pharmacological inhibition of NFkB, and low doses of the NFkB inhibitors, CBL0137, and emetine, efficiently reduced the CSC and the levels of SIRT1, increasing histone acetylation. The NFkB inhibitors decreased stemness potential, clonogenicity, migration, and invasion of cisplatin-resistant cell lines. The administration of the emetine significantly reduced the tumor growth of cisplatin-resistant xenograft models, decreasing NFkB and SIRT1, increasing histone acetylation, and decreasing CSC. TNF-alpha/NFkB/SIRT1 signaling regulates the epigenetic machinery by modulating histone acetylation, CSC, and aggressiveness of cisplatin-resistant OSCC and the NFkB inhibition is a potential strategy to treat chemoresistant OSCC.     

Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines

Resistance of oral squamous cell carcinomas (OSCC) to conventional chemotherapy or radiation therapy might be due to cancer stem cells (CSCs). The development of novel anticancer drugs requires a simple method for the enrichment of CSCs. CSCs can be enriched from OSCC cell lines, for example, after cultivation in serum-free cell culture medium (SFM). In our study, we analyzed four OSCC cell lines for the presence of CSCs. CSC-like cells could not be enriched with SFM. However, cell lines obtained from holoclone colonies showed CSC-like properties such as a reduced rate of cell proliferation and a reduced sensitivity to Paclitaxel in comparison to cells from the parental lineage. Moreover, these cell lines differentially expressed the CSC-marker CD133, which is also upregulated in OSCC tissues. Interestingly, CD133⁺ cells in OSCC tissues expressed little to no Ki67, the cell proliferation marker that also indicates reduced drug sensitivity. Our study shows a method for the isolation of CSC-like cell lines from OSCC cell lines. These CSC-like cell lines could be new targets for the development of anticancer drugs under in vitro conditions.     

Radiation responses of cancer stem cells

Recent experimental evidence indicates that many solid cancers have a hierarchical organization structure with a subpopulation of cancer stem cells (CSCs). The ability to identify CSCs prospectively now allows for testing the responses of CSCs to treatment modalities like radiation therapy. Initial studies have found CSCs in glioma and breast cancer relatively resistant to ionizing radiation and possible mechanisms behind this resistance have been explored. This review summarizes the landmark publications in this young field with an emphasis on the radiation responses of CSCs. The existence of CSCs in solid cancers place restrictions on the interpretation of many radiobiological observations, while explaining others. The fact that these cells may be a relatively quiescent subpopulation that are metabolically distinct from the other cells in the tumor has implications for both imaging and therapy of cancer. This is particularly true for biological targeting of cancer for enhanced radiotherapeutic benefit, which must consider whether the unique properties of this subpopulation allow it to avoid such therapies. J. Cell. Biochem. 108: 339–342, 2009. © 2009 Wiley‐Liss, Inc.     

CD276 expression enables squamous cell carcinoma stem cells to evade immune surveillance

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IGHMBP2过表达促进食管鳞癌细胞的侵袭和迁移

IGHMBP2(Immunoglobulin mu binding protein 2)基因编码一种解旋酶,参与DNA的复制和修复,并且作为转录调节因子在基因转录中发挥重要作用。IGHMBP2基因定位于11q13.2,该染色体区段在食管鳞癌中扩增频率较高。为了探讨IGHMBP2基因在食管鳞癌中的扩增情况及其在食管鳞癌中的作用,文章对本实验室前期报道的59例食管鳞癌原发肿瘤array-CGH数据进行分析,结果显示IGHMBP2基因扩增频率为28.9%(17/59)。进一步利用荧光原位杂交(FISH)和Western blot技术,发现食管鳞癌细胞系KYSE30、KYSE180、KYSE510和KYSE150中存在IGHMBP2基因扩增/增益以及蛋白高表达。敲降IGHMBP2后,KYSE30和KYSE150细胞的侵袭迁移能力明显降低(P<0.001),侵袭迁移相关蛋白E-cadherin的表达水平升高;敲降后转染IGHMBP2质粒,回复其蛋白表达后,细胞的侵袭迁移能力又得以恢复(P<0.01)。上述结果表明,IGHMBP2过表达可能通过降低E-cadherin的表达从而增强食管鳞癌细胞的侵袭迁移能力。     

KITENIN increases invasion and migration of mouse squamous cancer cells and promotes pulmonary metastasis in a mouse squamous tumor model

KAI1 C-terminal interacting tetraspanin (KITENIN) is reported to promote metastasis in mouse colon cancer models. We investigated the role of KITENIN on the progression of squamous cell carcinoma (SCC). In a preliminary clinical study using resected tissues from head and neck SCC patients, KITENIN was highly expressed in tumors and metastatic lymph nodes, while KAI1 was more increased in adjacent mucosa than in tumor. KITENIN-transfected mouse squamous cancer (SCC VII/KITENIN) cells showed significantly higher invasion, migration, and proliferation than empty vector-transfected cells. In syngeneic mouse squamous tumor models, more increased tumor volume and enhanced lung metastasis were found in SCC VII/KITENIN cells-injected mice. Thus, KITENIN increases invasion and migration of squamous cancer cells and thereby promotes distant metastasis in mouse squamous tumor models.     

The promise of stem cell markers in the diagnosis and therapy of epithelial dysplasia and oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is the most common type of head and neck cancer. Epithelial dysplasia is often initiated in the cells and cell nuclei adjacent to the epithelial cell membrane. Reduced cell–cell adhesions enable cancer cells to detach from the tumor and disseminate to other organs. The mutations in epithelial dysplasia markers such as E‐cadherin and epithelial cell adhesion molecules (CD326) can lead to proliferation, growth and survival of the tumor cells and persistence of numerous malignancies that play a key role in epithelial dysplasia of OSCC. Accordingly, these genes can be considered prognostic markers or potential therapeutic targets for the tailored management of patients with OSCC. The gene expression profile of OSCC stem cells indicates a differential pattern that facilitates establishing a cell signature. Owing to the highly tumorigenic behavior of cancer stem cells and the role of these cells in tumor differentiation, treatment resistance, relapse, and metastasis, we reviewed the role of stem cell markers in epithelial dysplasia and OSCC.     

SOD2 is a C-myc target gene that promotes the migration and invasion of tongue squamous cell carcinoma involving cancer stem-like cells

Our previous studies revealed that manganese superoxide dismutase (SOD2) contributes to the migration and invasion of tongue squamous cell carcinoma (TSCC). The purpose of the current study was to further clarify the mechanisms of SOD2 in the migration and invasion of TSCC. Side population (SP) cells were used as cancer stem-like cells and further assessed by sphere and colony formation assays, and the expression of stem cell markers (Bmi1, Nanog and ABCG2). We found that UM1 cells (TSCC cells with increased SOD2 expression, migration and invasion abilities) possessed a higher proportion of SP cells, sphere and colony formation, and expressed a higher level of stem cell markers compared to UM2 cells (reduced SOD2 expression, migration and invasion abilities). SOD2 expression as well as migration and invasion abilities were enhanced in SP cells compared to non-SP cells. Knockdown of SOD2 in UM1 cells or SP cells inhibited the migration and invasion abilities, reduced sphere and colony formation, and the expression of stem cell markers. Direct binding of the C-myc protein to the SOD2 promoter was demonstrated by chromatin immunoprecipitation and luciferase assays. Knockdown of C-myc in UM1 cells inhibited SOD2 expression as well as migration and invasion abilities. Our results indicate that cancer stem-like cells play an important role in the migration and invasion of TSCC. SOD2 is a direct target gene of C-myc and C-myc-SOD2-mediated migration and invasion of TSCC involve cancer stem-like cells.     

Tenascin-C and carcinoma cell invasion in oral and urinary bladder cancer

Carcinoma invasion is a complex process regulated by genetic and epigenetic factors as well. A relevant supportive condition for cancer cell migration is the reorganization of the extracellular matrix (ECM), which is realized in an orchestrated multicellular manner including carcinoma cells and stromal fibroblasts. An important key player in the process of ECM reorganization is Tenascin-C (Tn-C). The molecule occurs as different isoforms generated by alternative splicing and de novo glycosylation. Large variants of Tn-C are abundantly re-expressed in the invasive front of many carcinoma types. A special role for initiating migration and accompanied epithelial to mesenchymal transition has been suggested. Here, we review the current knowledge concerning the tumor biological importance of Tn-C, the synthesis and alternative splicing during the invasive process in general, and give an overview on the impact of Tn-C in urothelial carcinoma of the urinary bladder (UBC) and oral squamous cell carcinoma (OSCC).     

Over-expression of Oct4 in human esophageal squamous cell carcinoma

The Octamer 4 gene (Oct4) is a master pluripotency controller that has been detected in several types of tumors. Here, we examine the expression of Oct4 in human esophageal squamous cell carcinoma (ESCC). We found that punctate Oct4 protein was expressed in most (93.7%) ESCC samples but it was not observed in esophageal mucosa. Some ESCC cells had the capacity to form tumorospheres; those with an Oct4⁺-rich cell phenotype had increased proliferation and Oct4 mRNA levels compared to those of differentiated cells in culture or xenograft tumors. The over-expression of Oct4 in ESCCs suggests that it is a potential target for ESCC therapy. Oct4 could be a useful tumor marker in an immunohistochemical panel designed to differentiate between ESCC and esophageal mucosa. Expression of Oct4 in tumorospheres might indicate the presence of a population of ECSCs and its expression in xenograft tumors suggests that Oct4 is also associated with tumor metastasis.     

TUSC3 inhibits cell proliferation and invasion in cervical squamous cell carcinoma via suppression of the AKT signalling pathway

The decreased expression of tumour suppressor candidate 3 (TUSC3) is associated with proliferation in several types of cancer, leading to an unfavourable prognosis. The present study aimed to assess the cellular and molecular function of TUSC3 in patients with cervical squamous cell carcinoma (CSCC). Levels of mRNA expressions of TUSC3 were analysed in CSCC tissues and six cell lines using qRT‐PCR. Immunohistochemistry(IHC) was used to evaluate the protein expression level of TUSC3 in four paired specimens, 220 paraffin‐embedded CSCC specimens and 60 cases of normal cervical tissues(NCTs), respectively. Short hairpin RNA interference was employed for TUSC3 knockdown. Cell proliferation, migration and invasion were evaluated using growth curve, MTT assay, wound healing, transwell assay and xenograft tumour model, respectively. The results demonstrated that TUSC3 mRNA and protein expression levels were downregulated in CSCC samples. Multivariate and univariate analyses indicated that TUSC3 was an independent prognostic factor for patients with CSCC. Decreased TUSC3 expression levels were significantly associated with proliferation and an aggressive phenotype of cervical cancer cells both in vitro and in vivo. Moreover, the knockdown of TUSC3 promoted migration and invasion of cancer cells, while the increased expression of TUSC3 exhibited the opposite effects. The downregulation of TUSC3 facilitated proliferation and invasion of CSCC cells through the activation of the AKT signalling pathway. Our data demonstrated that the downregulation of TUSC3 promoted CSCC cell metastasis via the AKT signalling pathway. Therefore, TUSC3 may serve as a novel prognostic marker and potential target for CSCC.     

Immunohistochemical method identifies lymphovascular invasion in a majority of oral squamous cell carcinomas and discriminates between blood and lymphatic vessel invasion.

Tumor invasion into blood and/or lymphatic channels is an important component of cancer staging and prognosis. Standard pathological methods do not provide sufficient contrast to discriminate between invasion into each type of vessel and are complicated by tissue retraction artifacts. We evaluated the ability of a triple-stain immunohistochemical method, combining cytokeratin, CD34, and podoplanin stains in a single section, to distinguish blood from lymphatic vascular invasion in oral squamous cell carcinoma and confirmed its results using multispectral analysis. The triple-stain method was significantly more sensitive in detecting invasive events than the standard hematoxylin and eosin staining method and easily discriminated between blood and lymphatic vessel invasion. Invasive events were present in blood and/or lymphatic vessels in the majority of patients with and without presentation of lymph node metastasis, indicating that vessel invasion in this cancer model is common and is not a rate-limiting step for lymph node metastasis.     

Skin squamous cell carcinoma propagating cells increase with tumour progression and invasiveness

Cancer stem cells have been described in various cancers including squamous tumours of the skin by their ability to reform secondary tumours upon transplantation into immunodeficient mice. Here, we used transplantation of limiting dilution of different populations of FACS‐isolated tumour cells from four distinct mouse models of squamous skin tumours to investigate the frequency of tumour propagating cells (TPCs) at different stages of tumour progression. We found that benign papillomas, despite growing rapidly in vivo and being clonogenic in vitro, reformed secondary tumours upon transplantation at very low frequency and only when tumour cells were co‐transplanted together with tumour‐associated fibroblasts or endothelial cells. In two models of skin squamous cell carcinoma (SCC), TPCs increased with tumour invasiveness. Interestingly, the frequency of TPCs increased in CD34^HI but not in CD34^LO SCC cells with serial transplantations, while the two populations initially gave rise to secondary tumours with the same frequency. Our results illustrate the progressive increase of squamous skin TPCs with tumour progression and invasiveness and reveal that serial transplantation may be required to define the long‐term renewal potential of TPCs.     

Aldehyde dehydrogenase 1 is a putative marker for cancer stem cells in head and neck squamous cancer

Aldehyde dehydrogenase 1 (ALDH1) has been considered to be a marker for cancer stem cells. However, the role of ALDH1 in head and neck squamous cell carcinoma (HNSCC) has yet to be determined. In this study, we isolated ALDH1-positive cells from HNSCC patients and showed that these HNSCC-ALDH1⁺ cells displayed radioresistance and represented a reservoir for generating tumors. Based on microarray findings, the results of Western blotting and immunofluorescent assays further confirmed that ALDH1⁺-lineage cells showed evidence of having epithelial-mesenchymal transition (EMT) shifting and endogenously co-expressed Snail. Furthermore, the knockdown of Snail expression significantly decreased the expression of ALDH1, inhibited cancer stem-like properties, and blocked the tumorigenic abilities of CD44⁺CD24⁻ALDH1⁺ cells. Finally, in a xenotransplanted tumorigenicity study, we confirmed that the treatment effect of chemoradiotherapy for ALDH1⁺ could be improved by Snail siRNA. In summary, it is likely that ALDH1 is a specific marker for the cancer stem-like cells of HNSCC.     

Ezrin Ser66 phosphorylation regulates invasion and metastasis of esophageal squamous cell carcinoma cells by mediating filopodia formation

BackgroundEzrin, links the plasma membrane to the actin cytoskeleton, and plays an important role in the development and progression of human esophageal squamous cell carcinoma (ESCC). However, the roles of ezrin S66 phosphorylation in tumorigenesis of ESCC remain unclear.MethodsDistribution of ezrin in membrane and cytosol fractions was examined by analysis of detergent-soluble/-insoluble fractions and cytosol/membrane fractionation. Both immunofluorescence and live imaging were used to explore the role of ezrin S66 phosphorylation in the behavior of ezrin and actin in cell filopodia. Cell proliferation, migration and invasion of ESCC cells were investigated by proliferation and migration assays, respectively. Tumorigenesis, local invasion and metastasis were assessed in a nude mouse model of regional lymph node metastasis.ResultsEzrin S66 phosphorylation enhanced the recruitment of ezrin to the membrane in ESCC cells. Additionally, non-phosphorylatable ezrin (S66A) significantly prevented filopodia formation, as well as caused a reduction in the number, length and lifetime of filopodia. Moreover, functional experiments revealed that expression of non-phosphorylatable ezrin (S66A) markedly suppressed migration and invasion but not proliferation of ESCC cells in vitro, and attenuated local invasion and regional lymph node metastasis, but not primary tumor growth of ESCC cells in vivo.ConclusionEzrin S66 phosphorylation enhances filopodia formation, contributing to the regulation of invasion and metastasis of esophageal squamous cell carcinoma cells.     

The molecular markers of cancer stem cells in head and neck tumors

Head and neck cancer (HNC) is the six most common malignancy worldwide leading to more than 350,000 deaths annually. Despite recent advances in treatment modalities for these patients, there has been only a slight improvement of prognosis. As cancer stem cells (CSCs) have been implicated in tumor cell survival, progression, and response to therapy, the identification of this tumor subpopulation would have important therapeutic and prognostic implications. In this structured appraisal of the literature, Embase, PubMed, and Ovid were searched for publications that investigated CSC markers of HNC in humans. The search was conducted under the PRISMA guidelines with clear inclusion and exclusion criteria for articles published in the last two decades. The review process resulted in the identification of some key CSC-associated molecules such as CD44, ALDH1, CD133, Oct3/4, Nanog, and Sox2, although a single common CSC sorting marker could not be found. These biomarkers were identified in a range of HNCs but the most common one was squamous cell carcinoma (SCC), predominantly oral SCC. Patient cohorts were of variable size (3–195 individuals) and the most common technique used for detection was immunohistochemistry. Some of the molecules were associated with poor prognosis and may be able to inform the choice of appropriate treatment for these patients.