Wen-Liang Lo

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.

Outcomes of oral squamous cell carcinoma in Taiwan after surgical therapy: factors affecting survival.

PURPOSE The study goal was to determine which clinical features correlated with 5-year survival in patients surgically treated for oral squamous cell carcinoma (OSCC) in Taiwan. PATIENTS AND METHODS The records of 378 OSCC patients surgically treated with or without chemotherapy and radiotherapy were reviewed retrospectively. Their 5-year survival in relation to age, gender, tumor site, lymph node involvement, presence of distant metastasis, staging, differentiation, and risk factors, including betel quid (BQ) chewing, cigarette smoking, and alcohol consumption, was analyzed. RESULTS The majority of the patients were men (male-to-female ratio, 5.87:1) with the mean age of 57.1 +/-11.7 years. Tumors occurred mainly at the buccal mucosa (BM) (100 of 378, 26.5%), gingiva (105 of 378, 27.8%), and tongue (103 of 378, 27.2%). Neck nodal metastasis occurred frequently at the floor of the mouth (in >60% of cases), followed by the gingiva (45.7%), buccal mucosa (34%), and tongue (20.4%), whereas early distant metastasis was rare (5.3%). There were 104 (27.5%) stage 1, 96 (25.4%) stage 2, 98 (25.9%) stage 3, and 80 (21.2%) stage 4 patients. OSCC at the BM and gingiva was most (and at the tongue least) associated with risk factors of BQ use and smoking. The 5-year survival was 75%, 65.6%, 49%, and 30% for patients with stage I, II, III, and IV, respectively. The size, nodal involvement, distant metastasis, staging, differentiation, and BQ use significantly affected the survival (P <.05, Kaplan-Meier analysis). BQ use also correlated most significantly with the younger age of occurrence of OSCC patients. CONCLUSIONS Our data suggest that early treatment is the key to increasing the survival of OSCC patients. Periodic screening of high-risk populations for OSCC represents an urgent need in Taiwan.

MicroRNA-200c attenuates tumour growth and metastasis of presumptive head and neck squamous cell carcinoma stem cells†

MicroRNA‐200c (miR200c) is emerging as an important regulator of tumourigenicity and cancer metastasis with a strong capacity for inducing epithelial–mesenchymal transitions. However, the role of miR200c in head and neck squamous cell carcinoma (HNSCC) and HNSCC‐associated cancer stem cells (HNSCC‐CSCs) is unknown. In this study, the expression of miR200c in the regional metastatic lymph node of HNSCC tissues was significantly decreased, but BMI1 expression was increased as compared to parental tumours. Importantly, site‐directed mutagenesis with a luciferase reporter assay showed that miR200c targeted the 3′ UTR of BMI1 in HNSCC cells. Isolated HNSCC‐derived ALDH1+/CD44+ cells displayed CSC‐like tumour initiating and radio‐resistant properties. The expression levels of miR200c were significantly down‐regulated while BMI1 was increased in HNSCC‐ALDH1+/CD44+ compared to the other subsets of HNSCC cells. Furthermore, increased miR200c expression or knockdown of BMI1 could significantly inhibit the malignant CSC‐like properties of ALDH1+/CD44+ cells. miR200c over‐expression further down‐regulated the expressions of ZEB1, Snail and N‐cadherin, but up‐regulated E‐cadherin expression in ALDH1+/CD44+ cells. Finally, a xenotransplantion study confirmed that over‐expression of miR200c or BMI1 knockdown effectively inhibited the lung metastatic ability and prolonged the survival rate of ALDH1+/CD44+‐transplanted mice. In summary, miR200c negatively modulates the expression of BMI1 but also significantly inhibits the metastatic capability of epithelial–mesenchymal transitions in malignant HNSCC by reducing the expression of BMI1/ZEB1. Restoration of miR200c in HNSCC and CSCs may be a promising therapeutic approach. Copyright

MicroRNA let-7a represses chemoresistance and tumourigenicity in head and neck cancer via stem-like properties ablation

Head and neck cancer (HNC) is a prevalent cancer worldwide. Let-7 has been shown to function as a tumour suppressor by regulating multiple oncogenic signalling pathways. However, the role of let-7 in head and neck cancer (HNC) and in HNC-associated tumour initiating cells (TIC) remains unclear. In this study, we first demonstrated that let-7a expression was significantly decreased but that Nanog/Oct4 expression was increased in HNC tissues as compared to adjacent normal cells. Expression of let-7a in recurrent HNC tissue and in regional metastatic lymph nodes of HNC patients was also significantly decreased, but Nanog/Oct4 expression was increased as compared to the expression levels in the parental tumours. Consistently, the stemness genes were significantly up-regulated and let-7a was down-regulated in HNC-ALDH1(+) cells relative to HNC-ALDH1(-) cells. Furthermore, lentiviral-mediated let-7a overexpression could significantly inhibit the stemness signature and the chemoresistant abilities of HNC-ALDH1(+) cells. Most importantly, overexpression of let-7 or knockdown of Nanog in ALDH1(+) cells effectively blocked tumour metastasis and significantly prolonged survival time in ALDH1(+)-transplanted immunocompromised mice. Overall, restoration of let-7a in HNC and HNC-TIC may be a new approach for the therapeutic treatment of HNC in the future. These results show that let-7a negatively modulates the expression of stemness genes and plays a role as a tumour suppressor in HNC by eliminating the putative HNC-TIC population.

Inhibition of tumorigenicity and enhancement of radiochemosensitivity in head and neck squamous cell cancer-derived ALDH1-positive cells by knockdown of Bmi-1

Bmi-1, a member of the Polycomb family of transcriptional repressors, is essential for maintaining the self-renewal abilities of adult stem cells. Bmi-1 has been demonstrated to play a role in tumorigenesis in head and neck squamous cell carcinomas (HNSCCs). A recent study has further suggested that ALDH1 may be considered to be a putative marker for HNSCC-derived cancer stem cells. However, the role that Bmi-1 plays in HNSCC-derived ALDH1-positive cells (HNSCC-ALDH1(+)) has yet to be determined. In this study, we demonstrated that HNSCC-ALDH1(+) cells possess tumor initiating properties, are capable of self-renewal, and express higher levels of Bmi-1 as compared to HNSCC-ALDH1(-) cells. To further explore the functional role of Bmi-1 in HNSCC-ALDH1(+) cells, we used a lentiviral vector expressing shRNA to knock down Bmi-1 expression (sh-Bmi-1) in HNSCC-ALDH1(+) cells. Silencing of Bmi-1 significantly enhanced the sensitivity of HNSCC-ALDH1(+) cells to chemoradiation and increased the degree of chemoradiation-mediated apoptosis that occurred. Importantly, knockdown of Bmi-1 increased the effectiveness of radiotherapy and led to the inhibition of tumor growth in nude mice transplanted with HNSCC-ALDH1(+) cells. Kaplan-Meier survival analysis indicated that the mean survival rate of HNSCC-ALDH1(+) tumor-bearing immunocompromised mice treated with radiotherapy was significantly improved by treatment with sh-Bmi-1 as well. In summary, these results suggest that Bmi-1 is a potential target for increasing the sensitivity of HNSCC cancer stem cells to chemoradiotherapy.

Bmi-1 Regulates Snail Expression and Promotes Metastasis Ability in Head and Neck Squamous Cancer-Derived ALDH1 Positive Cells

Recent studies suggest that ALDH1 is a putative marker for HNSCC-derived cancer stem cells. However, the regulation mechanisms that maintain the stemness and metastatic capability of HNSCC-ALDH1+ cells remain unclear. Initially, HNSCC-ALDH1+ cells from HNSCC patient showed cancer stemness properties, and high expression of Bmi1 and Snail. Functionally, tumorigenic properties of HNSCC-ALDH1+ cells could be downregulated by knockdown of Bmi-1. Overexpression of Bmi-1 altered in expression property ALDH1− cells to that of ALDH1+ cells. Furthermore, knockdown of Bmi-1 enhanced the radiosensitivity of radiation-treated HNSCC-ALDH1+ cells. Moreover, overexpression of Bmi-1 in HNSCC-ALDH1− cells increased tumor volume and number of pulmonary metastatic lesions by xenotransplant assay. Importantly, knock-down of Bmi1 in HNSCC-ALDH1+ cells significantly decreased distant metastases in the lungs. Clinically, coexpression of Bmi-1/Snail/ALDH1 predicted the worst prognosis in HNSCC patients. Collectively, our data suggested that Bmi-1 plays a key role in regulating Snail expression and cancer stemness properties of HNSCC-ALDH1+ cells.

Epithelial-mesenchymal transition transcription factor ZEB1/ZEB2 co-expression predicts poor prognosis and maintains tumor-initiating properties in head and neck cancer.

OBJECTIVES Both epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) properties may be involved in metastasis, which contributes to the high mortality rate of patients with head and neck cancers (HNCs). However, the mechanisms through which the EMT transcription factors ZEB1 and ZEB2 regulate HNC are still unclear. METHODS Tumor initiating capability of HNC-CH133(+) cells with ZEB1/2 knockdown or co-overexpression was presented in vitro and in vivo. RESULTS In the present study, we demonstrated that ZEB1/ZEB2 expression was significantly increased in HNC-CD133(+) CSC-like cells compared with HNC-CD133(-) cells. The small interfering RNA (siRNA)-mediated co-knockdown of ZEB1 and ZEB2 (siZEB1/2) in HNC-CH133(+) cells suppressed their CSC-like properties, including self-renewal ability, the expression of stemness markers, and drug resistance. In contrast, the co-overexpression of ZEB1/ZEB2 in HNC-CD133(-) cells enhanced their sphere-forming ability and increased the percentage of CD44-positive cells and side population cells. In vivo studies showed that the delivery of siZEB1/2 to xenograft tumors in nude mice reduced tumor growth and the rate of distant metastasis. In clinical samples, the levels of ZEB1/ZEB2 expression were low in local lesions but high in metastatic lymph nodes in HNC tissues. Patients with tumors that co-expressed ZEB1(high) and ZEB2(high) had especially poor survival rates. CONCLUSION Therapies targeting ZEB1/ZEB2 in HNC-CD133(+) cells may provide a new approach for HNC therapy in the future.

Histopathological factors affecting nodal metastasis in tongue cancer: analysis of 94 patients in Taiwan

The overall prognosis for tongue cancer patients in Taiwan is unpredictable, even when patients are treated following the guidelines according to TNM stages. In order to determine the optimal treatment modality for tongue cancer in Taiwan the authors aimed to correlate histopathological parameters with neck nodal metastasis. A retrospective analysis of 94 patients with different stages of tongue cancer treated in the Taipei Veterans General Hospital was performed. All 94 patients were clinically diagnosed with stage I-IV tongue cancer before surgery and received primary tumor-wide excision and neck dissection. There were 42 (45%) patients with nodal metastasis. Univariate analysis revealed that cases of tongue cancer with moderate or poor differentiation, an invasion depth more than 3mm and positive perineural invasion or lymphovascular permeation at the time of presentation may be subject to a higher incidence of neck nodal metastasis. An elective neck dissection or neck treatment should be considered if these histopathological risk factors are present. Cases of tongue cancer with these risk factors also warrant close follow-up after surgery.

Synergistic effects of carboxymethyl-hexanoyl chitosan, cationic polyurethane-short branch PEI in miR122 gene delivery: accelerated differentiation of iPSCs into mature hepatocyte-like cells and improved stem cell therapy in a hepatic failure model.

MicroRNA122 (miR122), a liver-specific microRNA, plays critical roles in homeostatic regulation and hepatic-specific differentiation. Induced pluripotent stem cells (iPSCs) have promising potential in regenerative medicine, but it remains unknown whether non-viral vector-mediated miR122 delivery can enhance the differentiation of iPSCs into hepatocyte-like cells (iPSC-Heps) and rescue thioacetamide-induced acute hepatic failure (AHF) in vivo. In this study, we demonstrated that embedment of miR122 complexed with polyurethane-graft-short-branch polyethylenimine copolymer (PU-PEI) in nanostructured amphiphatic carboxymethyl-hexanoyl chitosan (CHC) led to dramatically enhanced miR122 delivery into human dental pulp-derived iPSCs (DP-iPSCs) and facilitated these DP-iPSCs to differentiate into iPSC-Heps (miR122-iPSC-Heps) with mature hepatocyte functions. Microarray and bioinformatics analysis further indicated that CHC/PU-PEI-miR122 promoted the gene-signature pattern of DP-iPSCs to shift into a liver-specific pattern. Furthermore, intrahepatic delivery of miR122-iPSC-Heps, but not miR-Scr-iPSC-Heps, improved liver functions and rescued recipient survival, and CHC-mediated delivery showed a better efficacy than that using phosphate buffered saline as a delivery vehicle. In addition, these transplanted miR122-iPSC-Heps remained viable and could produce circulatory albumin for 4 months. Taken together, our findings demonstrate that non-viral delivery of miR122 shortens the time of iPSC differentiation into hepatocytes and the delivery of miR122-iPSC-Heps using CHC as a vehicle exhibited promising hepatoprotective efficacy in vivo. miR122-iPSC-Heps may represent a feasible cell source and provide an efficient and alternative strategy for hepatic regeneration in AHF.

Network Biology of Tumor Stem-like Cells Identified a Regulatory Role of CBX5 in Lung Cancer

Mounting evidence links cancers possessing stem-like properties with worse prognosis. Network biology with signal processing mechanics was explored here using expression profiles of a panel of tumor stem-like cells (TSLCs). The profiles were compared to their parental tumor cells (PTCs) and the human embryonic stem cells (hESCs), for the identification of gene chromobox homolog 5, CBX5, as a potential target for lung cancer. CBX5 was found to regulate the stem-like properties of lung TSLCs and was predictive of lung cancer prognosis. The investigation was facilitated by finding target genes based on modeling epistatic signaling mechanics via a predictive and scalable network-based survival model. Topologically-weighted measurements of CBX5 were synchronized with those of BIRC5, DNMT1, E2F1, ESR1, MLH1, MSH2, RB1, SMAD1 and TAF5. We validated our findings in another Taiwanese lung cancer cohort, as well as in knockdown experiments using sh-CBX5 RNAi both in vitro and in vivo.