Se Yeong Oh

Cancer stem cell traits in squamospheres derived from primary head and neck squamous cell carcinomas

A subpopulation of cancer stem cells (CSCs), but not the majority of non-tumorigenic cancer cells, in a variety of human malignancies plays a critical role in cancer cell proliferation, invasion, metastasis, and tumor recurrence post-therapies. We report the isolation of sphere-forming cells (squamospheres) from primary head and neck squamous cell carcinomas (HNSCCs), and characterization of their CSC properties. Squamospheres appeared within 2 weeks after seeding as single-dissociated cells obtained from primary HNSCC specimens in serum-free culture conditions. Real-time RT-PCR and immunocytochemistry assays revealed that a number of stem cell markers, including CK5, OCT4, SOX2, and nestin, were up-regulated in HNSCC-driven squamospheres. Fluorescence-activated cell sorting (FACS) analysis showed that squamospheres contain enriched side population cells compared to serum-induced differentiated squamosphere cells. Furthermore, HNSCC-driven squamospheres appeared to be chemoresistant to cisplatin, 5-fluorouracil (FU), paclitaxel and doxetaxel, and showed increased levels of ABCG2, one of the ATP-binding cassette (ABC) transporters. Of particular interest, in sharp contrast to subcutaneous injection of 1×10(6) differentiated squamosphere cells, as few as 100 squamosphere cells were able to give rise to tumors in nude mice. Altogether, we assert that primary HNSCC-driven squamospheres possess CSC properties, and its functional analysis may provide a novel tool for investigating the tumorigenic process of HNSCC.

Identification of a peptide that interacts with Nestin protein expressed in brain cancer stem cells

Glioma stem cells (GSCs) are presumably major culprits for brain tumor initiation, progression, and recurrence after conventional therapies. Thus, selective targeting and eradication of GSCs may provide a promising and effective therapeutic approach. Here, we isolated a GSC-targeting (GSCT) peptide that demonstrated selective binding affinity for many undifferentiated GSCs using in vitro phage display technology. This GSCT peptide binds to isotypes of Nestin proteins specifically expressed in GSCs, enabling it to target Nestin-positive cells in human glioblastoma tissues. In human glioblastoma tissue specimens, the fluorescence-conjugated GSCT peptide could visualize putative GSC populations, showing its possible use as a diagnostic agent. GSCT peptide is also internalized into undifferentiated GSCs specifically in vitro, and moreover, intravenously injected GSCT peptide effectively penetrated into tissues, specifically accumulated in gliomas that arise from subcutaneous and orthotopic implantation, and predominantly targeted Nestin-positive cells in these tumors. Thus, our GSCT peptide may be useful for the development of more promising therapeutic and diagnostic modalities that target GSCs in brain tumors.

CD44-negative cells in head and neck squamous carcinoma also have stem-cell like traits

CD44 is generally accepted as a surrogate marker for head and neck squamous carcinoma cancer stem cells (HNSC CSCs) and only CD44+ HNSC cells have tumour initiating capacity. However, a recent report suggested that CSCs themselves might be heterogenous due to various genetic alterations. Here, we compared in vitro stem-like cell characteristics, chemoresistance and in vivo tumour formation capacity of CD44+ and CD44- HNSC cells obtained from primary HNSC patient specimens. CD44- HNSC spheroid cells generated spheroid cells again after seeding of single-dissociated spheroid CD44- HNSC cells. Immunocytochemistry assays revealed that various stem cell markers, including octamer-binding transcription factor 4 (OCT4), sex determining region Y-box 2 (SOX2) and nestin were up-regulated in CD44- spheroid cells, similar to CD44+ spheroid cells. Furthermore, CD44- spheroid cells appeared to be chemoresistant to cisplatin and showed increased levels of ABCG2, similar to CD44+ spheroid cells. Of most interest, as few as 1000 CD44- spheroid cells were able to give rise to tumours in nude mice. The collective data indicate that the cell surface marker CD44 cannot be used as a selective marker of spheroid-forming, tumour-initiating or chemoresistant cell populations, and further indicate the limitation of current HNSC CSC identification methods using the CD44 cell surface marker.

Tumoral RANKL activates astrocytes that promote glioma cell invasion through cytokine signaling.

The invasiveness of glioblastoma is a major cause of poor prognosis and relapse. However, the molecular mechanism controlling glioma cell invasion is poorly understood. Here, we report that receptor activator of nuclear factor kappa-B (NFκB) ligand (RANKL) promotes glioma cell invasion in vivo, but not in vitro. Unlike the invasiveness under in vitro culture conditions, in vivo xenograft studies revealed that LN229 cells expressing high endogenous RANKL generated more invasive tumors than U87MG cells expressing relatively low endogenous RANKL. Consistently, RANKL-overexpressing U87MG resulted in invasive tumors, whereas RANKL-depleted LN229 generated rarely invasive tumors. We found that the number of activated astrocytes was markedly increased in the periphery of RANKL-high invasive tumors. RANKL activated astrocytes through NFκB signaling and these astrocytes in turn secreted various factors which regulate glioma cell invasion. Among them, transforming growth factor β (TGF-β) signaling was markedly increased in glioblastoma specimens and xenograft tumors expressing high levels of RANKL. These results indicate that RANKL contributes to glioma invasion by modulating the peripheral microenvironment of the tumor, and that targeting RANKL signaling has important implications for the prevention of highly invasive glioblastoma.

The ID1-CULLIN3 Axis Regulates Intracellular SHH and WNT Signaling in Glioblastoma Stem Cells.

Inhibitor of differentiation 1 (ID1) is highly expressed in glioblastoma stem cells (GSCs). However, the regulatory mechanism responsible for its role in GSCs is poorly understood. Here, we report that ID1 activates GSC proliferation, self-renewal, and tumorigenicity by suppressing CULLIN3 ubiquitin ligase. ID1 induces cell proliferation through increase of CYCLIN E, a target molecule of CULLIN3. ID1 overexpression or CULLIN3 knockdown confers GSC features and tumorigenicity to murine Ink4a/Arf-deficient astrocytes. Proteomics analysis revealed that CULLIN3 interacts with GLI2 and DVL2 and induces their degradation via ubiquitination. Consistent with ID1 knockdown or CULLIN3 overexpression in human GSCs, pharmacologically combined control of GLI2 and β-CATENIN effectively diminishes GSC properties. A ID1-high/CULLIN3-low expression signature correlates with a poor patient prognosis, supporting the clinical relevance of this signaling axis. Taken together, a loss of CULLIN3 represents a common signaling node for controlling the activity of intracellular WNT and SHH signaling pathways mediated by ID1.

Anticancer properties of extracts from opuntia humifusa against human cervical carcinoma cells

In this study, we found that the total polyphenol and ascorbic acid levels in the fruit of Opuntia humifusa are higher than those in other parts of the plant. We further hypothesized that antioxidants in O. humifusa might affect the growth or survival of cancer cells. Hexane extracts of seeds and ethyl acetate extracts of fruits and stems significantly suppressed the proliferation of HeLa cervical carcinoma cells, but did not affect the proliferation of normal human BJ fibroblasts. Additionally, the extracts of O. humifusa induced G1 phase arrest in HeLa cells. The O. humifusa extracts reduced the levels of G1 phase-associated cyclin D1, cyclin-dependent kinase 4 (Cdk4), and phosphorylated retinoblastoma proteins. Moreover, p21(WAF1/Cip1) and p53 expression significantly increased after treatment. We examined the effects of ethyl acetate extracts of O. humifusa fruit (OHF) on HeLa cells xenograft tumor growth. OHF treatment significantly reduced tumor volume and this decrease was correlated with decreased Cdk4 and cyclin D1 expression. Furthermore, flavonoids, trans Taxifolin, and dihydrokaempferol, were isolated from OHF. Thus, this extract may be a promising candidate for treating human cervical carcinoma.

Cellular characteristics of head and neck cancer stem cells in type IV collagen-coated adherent cultures.

Although head and neck squamous carcinoma cancer stem cells (HNSC-CSCs) can be enriched in serum-free suspension cultures, it is difficult to stably expand HNSC-CSC lines in suspension due to spontaneous apoptosis and differentiation. Here, we investigated whether HNSC-CSCs can be expanded without loss of stem cell properties by adherent culture methods. Cell culture plates were coated with type IV collagen, laminin, or fibronectin. We examined cancer stem cell traits of adherent HNSC-CSCs grown on these plates using immunocytochemistry for stem cell marker expression and analyses of chemo-resistance and xenograft tumorigenicity. We also assessed the growth rate, apoptosis rate, and gene transduction efficiency of adherent and suspended HNSC-CSCs. HNSC-CSCs grew much faster on type IV collagen-coated plates than in suspension. Adherent HNSC-CSCs expressed putative stem cell markers (OCT4 and CD44) and were chemo-resistant to various cytotoxic drugs (cisplatin, fluorouracil, paclitaxel, and docetaxel). Adherent HNSC-CSCs at the limiting dilution (1000 cells) produced tumors in nude mice. Adherent HNSC-CSCs also showed less spontaneous apoptotic cell death and were more competent to lentiviral transduction than suspended HNSC-CSCs. In conclusion, compared to suspension cultures, adherence on type IV collagen-coated culture plates provides better experimental conditions for HNSC-CSC expansion, which should facilitate various refined cellular studies.