Integrin alpha 6 as a stemness driver is a novel promising target for HPV (+) head and neck squamous cell carcinoma.

Although the incidence rates of head and neck squamous cell carcinoma (HNSCC) associated with human papilloma virus (HPV) infection have recently been on the rise, the underlying mechanism of its tumorigenesis remains largely unknown. Here, we investigated whether HNSCC cells with high expression of integrin alpha 6 (ITGα6), one of the HPV receptors, have a preference during HPV infection. In addition, we examined the gain or loss of function of the ITGα6 gene in HPV + ve HNSCC cells, as well as its prognostic value in patients with HNSCC. HPV pseudovirus was found to be more infective, with HNSCC cells featuring an overexpressed ITGα6 gene compared to the control cells. Overexpression and suppression of ITGα6 respectively increases and decreases stemness phenotypes of HPV + ve HNSCC cells. Furthermore, ITGα6 can regulate stemness by partially mediating AKT pathway in HPV + ve HNSCC cells. Finally, patients with HPV + ve HNSCC had a poor prognosis in cases of elevated ITGα6 expression; however, the expression levels of ITGα6 did not influence the survival rates of HPV-negative HNSCC patients. In conclusion, ITGα6 can serve as a potential therapeutic target for HPV + ve HNSCC cancer-like stem cells (CSCs).

Single clonal glandular stem cells derived from human parotid glands do not attain malignant phenotype during long-term in vitro culture.

Mesenchymal stem cells (MSCs) are being intensively investigated as future therapeutics for various human diseases. One of the most important challenges to the clinical application of MSCs is the possibility of malignant transformation during long-term in vitro culturing. However, there have been no reports on the tumorigenicity of salivary gland-derived MSCs following long-term in vitro culturing. Here, we isolated a single clonal glandular stem cells from human parotid gland stem cells (hpGSCs) using a modified sub-fractionation culturing method. The possibility of malignant transformation of these cells following long-term culturing was evaluated under in vitro and in vivo culture conditions. Single clonal glandular stem cells from the human parotid gland have unique multipotent MSCs traits. hpGSCs at passage 18 stained strongly for ß-galactosidase expression and the long-term culture of hpGSCs led to a reduction in telomerase activity. hpGSCs could not survive in a soft agar environment and did not cause tumor formation in a xenograft mouse model. In addition, the expression of salivary cancer-related oncogenes was not elevated in hpGSCs following the long-term culture. In conclusion, we demonstrated that there is no possibility of acquiring a malignant transformation during long-term in vitro cell expansion of hpGSCs.

Inhibitor of DNA Binding 2 (ID2): A Novel Marker for Lymph Node Metastasis in Head and Neck Squamous Cell Carcinoma.

BACKGROUND: Although aggressive invasion and sequential lymph node metastasis (LNM) significantly affect the prognosis of patients with head and neck squamous cell carcinoma (HNSCC), studies on identifying the factors that regulate this process remain scarce. This study found an inhibitor of DNA binding 2 (ID2) as a novel molecule involved in the regulation of invasion and LNM of HNSCC and further verified its functional role. METHODS: The study examined the translational significance between ID2 expression levels and the presence of LNM as well as the prognosis for 119 patients with HNSCC after treatment. In addition, in vitro and in vivo experiments were performed using ID2 gene-modulated HNSCC cell lines to determine the functional role of ID2 in the invasion and LNM of HNSCC. RESULTS: Elevated levels of ID2 expression were closely associated with the presence of LNM in 119 patients with HNSCC, resulting in a poor prognosis. Overexpression of ID2-induced invasion and LNM of HNSCC cells was observed in vitro and in vivo. By contrast, knockdown of the ID2 gene diminished invasion and LNM of HNSCC cells. In addition, the ID2 expression level increased the expression level of matrix metalloproteinase 1 (MMP1), a molecule downstream to ID2. Furthermore, silencing of MMP1 in ID2-overexpressed HNSCC cells rescued the elevated invasion and LNM capabilities of these cells, suggesting that ID2 enhances invasion and LNM partly via MMP1 activation. CONCLUSION: In the invasion and LNM of HNSCC, ID2 plays an important role by modulating MMP1 expression, suggesting ID2-MMP1 axis to be a novel alternative therapeutic target for invasion and LNM of HNSCC.

Wnt/ß-catenin/Slug pathway contributes to tumor invasion and lymph node metastasis in head and neck squamous cell carcinoma.

The canonical Wnt/ß-catenin pathway is involved in diverse cancer development mechanisms, such as proliferation, migration, and invasion. However, its role in head and neck squamous cell carcinoma (HNSCC) remains largely unknown. We investigated whether the canonical Wnt/ß-catenin signaling pathway acts as a controller of invasion and lymph node metastasis (LNM) in HNSCC. Loss of function experiments against the canonical Wnt/ß-catenin pathway were conducted to evaluate its invasive and metastatic role in HNSCC cells. Slug was evaluated as a downstream protein in canonical Wnt/ß-catenin-mediated invasion. In addition, canonical Wnt/ß-catenin and Slug expression levels were examined in 119 HNSCC tissue samples to study the relevance of these proteins in LNM and prognosis of patients post-treatment. In vitro suppression of ß-catenin expression led to decreased migration and invasion of HNSCC cells. Using an in vivo mouse orthotopic LNM model, a decrease in LNM was observed with mitigated ß-catenin expression. Slug expression upregulation mediates invasion and LNM by the canonical Wnt/ß-catenin pathway. Simultaneous expression of ß-catenin and Slug is the major predictive factor of LNM and survival rate in patients with HNSCC. In conclusion, the canonical Wnt/ß-catenin/Slug signaling axis significantly contributes to cancer cell invasion and LNM. Its blockade may be a treatment strategy for LNM and tumor recurrence in HNSCC.

Slug is a novel molecular target for head and neck squamous cell carcinoma stem-like cells.

BACKGROUND: The acquisition of stem-like phenotype is partly attributed to the induction of epithelial-mesenchymal transition (EMT). Thus, the activation of factors involved in EMT can be linked to cancer stem cell genesis. However, the underlying mechanisms in head and neck squamous cell carcinoma (HNSCC) remain largely unknown. Herein, we investigate whether slug, one of the major effectors of EMT, affects the stemness of HNSCC cells. METHODS: We performed in vitro experiments to determine whether slug gene manipulation can influence the stemness phenotypes, including the capacity for self-renewal, expression of putative stemness markers, chemoresistance, and invasion in HNSCC cells. Further, we identified whether Slug knockout attenuates tumorigenicity of HNSCC cells in vivo. Finally, we examined whether prognosis of HNSCC patients after curative treatment may be affected by the level of slug expression. RESULTS: Overexpression of slug promoted self-renewal of HNSCC cells via activation of sphere formation, the expression of stem cell markers, and induction of chemoresistance to cisplatin. Also, slug overexpression increased the migration and invasion of HNSCC cells in vitro and was mainly observed during the invasion in HNSCC xenograft mouse model. By contrast, slug expression knockdown abrogated their self-renewal capacity, stemness-associated gene expression, and cisplatin chemoresistance. Furthermore, high levels of slug expression correlated with poor prognosis of patients with HNSCC. CONCLUSION: Inhibition of slug expression may represent a novel therapeutic strategy targeting HNSCC stem-like cells.

Role of integrin ß1 as a biomarker of stemness in head and neck squamous cell carcinoma.

OBJECTIVES: Signaling between cancer stem cells (CSC) and their extracellular matrix has a crucial role in CSC progression and maintenance. However, mediators of this signaling pathway in head and neck squamous cell carcinoma (HNSCC) are largely unknown. Here, we explored whether integrin ß1, which is one of the key regulators of the communication between cells and their microenvironment, affected the stemness of HNSCC cells. MATERIALS AND METHODS: We examined self-renewal capacity, chemoresistance, and xenograft tumorigenicity after knockdown of integrin ß1 in primary HNSCC cells. In addition, we studied the role of focal adhesion kinase (FAK), an intracellular downstream molecule of integrin signaling, in influencing stemness of HNSCC. The relevance of Notch1 and integrin ß1 interactions in HNSCC cells was also examined. Finally, immunohistochemical analysis was carried out to test whether the coexpression of integrin ß1 and Notch1 in the samples from HNSCC patients correlated with their survival. RESULTS: Targeting integrin ß1 in HNSCC cells inhibited self-renewal, chemoresistance, and in vivo tumor-forming capacity. Treatment with an inhibitor of FAK decreased self-renewal capacities and expression of various putative stem cell markers (Oct4, Sox2, and Nanog) in a dose-dependent manner. Moreover, knockdown of integrin ß1 decreased the expression of Notch1 and its target genes (Hey1 and Hes1). Notably, HNSCC patients demonstrating simultaneous expression of integrin ß1 and Notch1 in their tissue samples had significantly worse survival rate. CONCLUSION: Integrin ß1/Notch1 axis has a significant role in the regulation of stemness in HNSCC.

Inhibitor of DNA binding 2 is a novel therapeutic target for stemness of head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinomas (HNSCCs) are highly lethal epithelial tumours containing self-renewal cancer stem cells (CSCs). CSCs in HNSCCs are strongly associated with tumour initiation, invasion, and chemoradiation resistance. However, the important factors regulating stemness in HNSCCs remain unclear. Here, we investigated the molecular roles and clinical significance of inhibitor of DNA binding 2 (Id2) protein to determine if it constitutes a novel therapeutic target for ablating HNSCC cells with stemness. METHODS: We performed in vitro and in vivo studies of Id2 function and its effects on stemness using HNSCC cells. We also examined whether Id2 expression could be used as a prognostic indicator through immunohistochemical staining of 119 human HNSCC tumours. RESULTS: Expression of Id2 was higher in HNSCC cells with stemness compared with differentiated HNSCC cells. Overexpression of Id2 increased proliferation, self-renewal, and expression of the putative stemness marker CD44 in HNSCC cells in vitro and in vivo. In contrast, silencing of Id2 using short hairpin RNA attenuated the stemness phenotype of HNSCC cells by reducing self-renewal, CD44 expression, cisplatin chemoresistance, and xenograft tumourigenicity. Most importantly, increased expression of Id2 was closely associated with poorer post-treatment survival rates in HNSCC patients. CONCLUSIONS: Inhibitor of DNA binding2 represents a novel and promising therapeutic target for treating and improving the clinical outcomes for patients with HNSCC.

C-Met pathway promotes self-renewal and tumorigenecity of head and neck squamous cell carcinoma stem-like cell.

OBJECTIVES: Increasing evidence indicates that a rare subpopulation of cancer cells, namely, cancer stem cells (CSCs), is the primary cause of tumorigenesis in some tumor types, including head and neck squamous cell carcinoma (HNSCC). Hepatocyte growth factor (HGF) and its receptor, c-Met, are involved in tongue development and carcinogenesis of HNSCC. Here, we investigated whether activation of HGF/c-Met signaling influences the stem cell traits of HNSCC CSC. MATERIALS AND METHODS: After HGF treatment, we assessed the sphere-forming capacity and stem cell marker expression in HNSCC stem-like cells compared with the respective control cells in vitro. In addition, we evaluated the effect of c-Met knockdown on the sphere-forming capacity, stem cell marker expression, and cisplatin chemosensitivity of HNSCC stem-like cells in vitro. Furthermore, we evaluated the inhibitory effect of c-Met knockdown on the capacity of HNSCC stem-like cells to initiate tumor growth in the orthotopic mouse model. RESULTS: HGF treatment promoted the sphere-forming capacity of HNSCC stem-like cells and increased the expression of stem cell markers such as Oct4, Sox2, and CD44. Transcriptional levels of c-Met was increased in cells with high aldehyde dehydrogenase (ALDH) 1 activity, a putative marker for HNSCC stem-like cells, compared to cells with low activity. In contrast, knockdown of c-Met attenuated the sphere-forming capacity and stem cell markers expression in HNSCC stem-like cells, and augmented cisplatin chemosensitivity by decreased side population cells and suppression of an ABCG2 transporter gene. Furthermore, knockdown of c-Met in HNSCC stem-like cells inhibits tumor formation of mice in a xenograft model and increases mice survival. CONCLUSION: Our results suggest that inhibition of the c-Met pathway could serve as a potent therapeutic strategy to target HNSCC stem-like cells.

Wnt/ß-catenin signalling maintains self-renewal and tumourigenicity of head and neck squamous cell carcinoma stem-like cells by activating Oct4.

Accumulating evidence suggests that a distinct subpopulation of cancer stem cells (CSCs) is responsible for tumour initiation and progression in head and neck squamous cell carcinoma (HNSCC). Wnt/ß-catenin signalling is essential for stem cell regulation and tumourigenesis, but its molecular mechanism in HNSCC CSCs remains unknown. We investigated whether Wnt/ß-catenin signalling regulates self-renewal and tumourigenicity of HNSCC stem-like cells in vitro and in vivo. Cytoplasmic/nuclear ß-catenin, a major effector of Wnt/ß-catenin signalling, was expressed in a subpopulation of tumour cells in primary HNSCC tissue but in none of normal head and neck tissues. Overexpression of ß-catenin increased proliferation of HNSCC cells and induced dedifferentiation of these cells to cells with stem-like features. Knockdown of ß-catenin in HNSCC stem-like cells blocked their self-renewal capacity, stemness-associated gene expression, chemoresistance, and in vivo tumourigenicity. Furthermore, ß-catenin directly regulates Oct4 transcription in HNSCC stem-like cells. In addition, the effect of shRNA-mediated repression of ß-catenin on CSC traits in HNSCC stem-like cells was reversed by overexpression of Oct4. In patients with HNSCC, higher levels of both cytoplasmic/nuclear ß-catenin and Oct4 correlated with the worst prognosis. These results suggest inhibition of Wnt/ß-catenin signalling as a novel therapeutic strategy for targeting HNSCC stem-like cells.

Three-week combination chemotherapy with S-1 and cisplatin as first-line treatment in patients with advanced gastric cancer: a retrospective study with 159 patients.

BACKGROUND: Doses and schedules of the combination of S-1 and cisplatin for the treatment of advanced gastric cancer (AGC) have not been standardized. We therefore evaluated the efficacy and feasibility of a 3-week schedule of S-1 and cisplatin in patients with AGC, as well as assessing factors prognostic of patient outcomes. METHODS: A total of 159 patients with AGC were treated with S-1 (40 mg/m(2) bid on days 1-14) and cisplatin (60 mg/m(2) IV on day 1) between January 2004 and December 2008. RESULTS: Median follow-up duration was 20.0 months (range, 11.4-48.5 months), during which time 129 patients (81.1%) died. Patients received a median 6 cycles of chemotherapy (range, 1-19 cycles). Among the 59 patients with measurable disease, 1 achieved a complete response (1.7%) and 24 (40.7%) had partial responses, giving an overall response rate of 42.4% (95% CI, 23.0-61.8%). The median progression-free survival (PFS) was 5.8 months (95% CI, 4.8-6.9 months), and the median overall survival (OS) was 11.3 months (95% CI, 9.6-13.0 months). Multivariate analysis showed that initial metastasis, bone metastasis, and liver metastasis were independent prognostic factors for reduced PFS, whereas poor performance status, initial metastasis, and bone metastasis were prognostic for reduced OS. Application of a previous prognostic model showed that observed PFS and OS survival curves for patients in various risk groups differed significantly (P < 0.001 each). CONCLUSIONS: A 3-week regimen of S-1 plus cisplatin was active and well tolerated as first-line treatment in patients with AGC. Disease status and bone metastasis were the most important prognostic factors.

A prognostic model in patients who receive chemotherapy for metastatic or recurrent gastric cancer: validation and comparison with previous models.

PURPOSE: To make up for the limitations of previous prognostic models, we developed and validated a model in patients with metastatic or recurrent gastric adenocarcinoma (AGC), and to compare with previous models. METHODS: A total of 2,805 patients received chemotherapy for AGC in Asan Medical Center between January 2000 and December 2008 and were randomly split into training and validation sets of 1,870 and 935 patients, respectively. A prognostic model was developed from the training set. RESULTS: The median follow-up duration was 26.5 months (range, 10.8-116.3), during which time 2,495 patients (88.9%) died. Eight factors associated with poor prognosis were identified by multivariate analysis: ECOG performance status ≥2 (2 points), no gastrectomy, peritoneal metastasis, bone metastasis (2 points), lung metastasis, alkaline phosphatase > 120 IU/l, albumin < 3.3 g/dL, and total bilirubin > 1.2 mg/dL. A prognostic model was developed by dividing patients into good (0-1 points), moderate (2-3), and poor (≥4) risk groups. The overall survival (OS) curves for three risk groups differed significantly for both the training and the validation sets (P < 0.001 each). In the training set, the median OS for the three risk groups was 14.0, 9.4, and 5.1 months, respectively. Application of three previous prognostic models to our validation set showed that the four models, including ours, had similar ability to predict survival outcomes (c-statistics, 0.5520-0.5836). CONCLUSION: Validation and comparison of prognostic models indicated that our model was as effective as the previous models to stratify the patients with AGC.