Cong-Fa Huang

CD163+ tumor-associated macrophages correlated with poor prognosis and cancer stem cells in oral squamous cell carcinoma.

Tumor-associated macrophages (TAMs) play an important role in the progression and prognostication of numerous cancers. However, the role and clinical significance of TAM markers in oral squamous cell carcinoma (OSCC) has not been elucidated. The present study was designed to investigate the correlation between the expression of TAM markers and pathological features in OSCC by tissue microarray. Tissue microarrays containing 16 normal oral mucosa, 6 oral epithelial dysplasia, and 43 OSCC specimens were studied by immunohistochemistry. We observed that the protein expression of the TAM markers CD68 and CD163 as well as the cancer stem cell (CSC) markers ALDH1, CD44, and SOX2 increased successively from the normal oral mucosa to OSCC. The expressions of CD68 and CD163 were significantly associated with lymph node status, and SOX2 was significantly correlated with pathological grade and lymph node status, whereas ALDH1 was correlated with tumor stage. Furthermore, CD68 was significantly correlated with CD163, SOX2, and ALDH1 (P < 0.05). Kaplan-Meier analysis revealed that OSCC patients overexpressing CD163 had significantly worse overall survival (P < 0.05). TAM markers are associated with cancer stem cell marker and OSCC overall survival, suggesting their potential prognostic value in OSCC.

PD-1 blockade attenuates immunosuppressive myeloid cells due to inhibition of CD47/SIRPα axis in HPV negative head and neck squamous cell carcinoma.

Myeloid-derived suppressor cells (MDSCs) and tumor associated macrophages (TAMs) play key roles in the tumor immune suppressive network and tumor progression. However, precise roles of programmed death-1 (PD-1) in immunological functions of MDSCs and TAMs in head and neck squamous cell carcinoma (HNSCC) have not been clearly elucidated. In the present study, we show that PD-1 and PD-L1 levels were significantly higher in human HNSCC specimen than in normal oral mucosa. MDSCs and TAMs were characterized in mice and human HNSCC specimen, correlated well with PD-1 and PD-L1 expression. αPD-1 treatment was well tolerated and significantly reduced tumor growth in the HNSCC mouse model along with significant reduction in MDSCs and TAMs in immune organs and tumors. Molecular analysis suggests a reduction in the CD47/SIRPα pathway by PD-1 blockade, which regulates MDSCs, TAMs, dendritic cell as well as effector T cells. Hence, these data identify that PD-1/PD-L1 axis is significantly increased in human and mouse HNSCC. Adoptive αPD-1 immunotherapy may provide a novel therapeutic approach to modulate the micro- and macro- environment in HNSCC.

Mammalian Target of Rapamycin Pathway Promotes Tumor-Induced Angiogenesis in Adenoid Cystic Carcinoma: Its Suppression by Isoliquiritigenin through Dual Activation of c-Jun NH2-Terminal Kinase and Inhibition of Extracellular Signal-Regulated Kinase

Tumor-induced angiogenesis is essential for invasive growth and hematogenous metastasis of adenoid cystic carcinoma (ACC), a highly aggressive neoplasm mostly occurring in salivary glands. Previous studies have indicated that strategies directed against angiogenesis will help develop new therapeutic agents for ACC. The Chinese folk medicine licorice has been used for years as a natural remedy for angiogenesis-related diseases. In this study, we examined the effects of isoliquiritigenin (ISL), a flavonoid isolated from licorice, on the growth and viability of ACC cells and observed a concentration-dependent (0–20 μM) inhibition of cell growth without cell death at 24 h. In a further mimic coculture study, ISL effectively suppressed the ability of ACC cells to induce in vitro proliferation, migration, and tube formation of human endothelial hybridoma (EAhy926) cells as well as ex vivo and in vivo angiogenesis, whereas it exerted no effect on EAhy926 cells when added directly or in the presence of vascular endothelial growth factor (VEGF). The data also showed that the specific suppression of tumor angiogenesis by ISL was caused by down-regulation of mammalian target of rapamycin (mTOR) pathway-dependent VEGF production by ACC cells, correlating with concurrent activation of c-Jun NH2-terminal kinase (JNK) and inhibition of extracellular signal-regulated kinase (ERK). Most importantly, ISL also significantly decreased microvessel density within xenograft tumors, associating with the reduction of VEGF production and suppression of the mTOR pathway coregulated by JNK and ERK, as revealed by immunohistochemical studies and clustering analysis. Taken together, our results highlight the fact that ISL is a novel inhibitor of tumor angiogenesis and possesses great therapeutic potential for ACC.

Correlation of ALDH1, CD44, OCT4 and SOX2 in tongue squamous cell carcinoma and their association with disease progression and prognosis.

BACKGROUND Recently, studies indicated that cancer stem cell plays a key role in cancer development and progression. However, the role of cancer stem cell has not been well elucidated in tongue squamous cell carcinoma (TSCC). The objective of this study was to investigate the relationships between the expressions of stem cell markers and the prognosis of TSCC. MATERIALS AND METHODS Immunohistochemistry was employed to analyse the protein expression levels of ALDH1, CD44, OCT4 and SOX2 in 66 TSCC tissue samples. The results were then evaluated semiquantitatively and compared with other clinicopathological variables. RESULTS Immunohistochemistry revealed that the ALDH1, CD44, OCT4 and SOX2 proteins were overexpressed in the 66 TSCC specimens used in this study. Spearmans correlation analysis showed that the expressions of ALDH1 and CD44 were significantly correlated with SOX2 except other proteins (P < 0.05) and that OCT4 and SOX2 were significantly related (P < 0.01). Kaplan-Meier analysis revealed that T category, node metastasis, TNM stage, differentiation and distant metastasis were associated with poor patient survival (P < 0.05). Multivariate Cox regression analysis demonstrated that SOX2, recurrence and distant metastasis were independent prognostic factors of overall survival in patients with TSCC. CONCLUSION Taken together, these data suggest that the stem cell markers ALDH1, CD44, OCT4 and SOX2 are closely related in TSCC, and the expression of SOX2 can be used as a prognostic indicator of TSCC.

Clinical significance of Keap1 and Nrf2 in oral squamous cell carcinoma.

Oxidative stress has been reported to play an important role in progression and prognostication in various kinds of cancers. However, the role and clinical significance of oxidative stress markers Keap1 and Nrf2 in oral squamous cell carcinoma (OSCC) has not been elucidated. This study aimed to investigate the correlation of oxidative stress markers Keap1 and Nrf2 expression and pathological features in OSCC by using tissue microarray. Tissue microarrays containing 17 normal oral mucosa, 7 oral epithelial dysplasia and 43 OSCC specimens were studied by immunohistochemistry. The association among these proteins and pathological features were analyzed. Expression of oxidative stress markers Keap1, Nrf2, and antioxidants PPIA, Prdx6, as well as CD147 was found to increase consecutively from normal oral mucosa to OSCC, and the Keap1, Nrf2, PPIA, Prdx6, CD147 expression in OSCC were significantly higher when compared to normal oral mucosa. Expression of Keap1, Nrf2 in tumors was not found to be significantly associated with T category, lymph node metastases, and pathological grade. Furthermore, we checked the relationship among these oxidative stress markers and found that Keap1 was significantly correlated with Nrf2, Prdx6 and CD147. Significant relationship between Nrf2 and Prdx6 was also detected. Finally, we found patients with overexpression of Keap1 and Nrf2 had not significantly worse overall survival by Kaplan–Meier analysis. These findings suggest that ROS markers are associated with carcinogenesis and progression of OSCC, which may have prognostic value and could be regarded as potential therapeutic targets in OSCC.

Association of increased ligand cyclophilin A and receptor CD147 with hypoxia, angiogenesis, metastasis and prognosis of tongue squamous cell carcinoma.

Huang C, Sun Z, Sun Y, Chen X, Zhu X, Fan C, Liu B, Zhao Y & Zhang W 
(2012) Histopathology 60, 793–803

Increased Expression of Lin28B Associates with Poor Prognosis in Patients with Oral Squamous Cell Carcinoma

Recent studies showed that incomplete cell reprogramming can transform cells into tumour-like cells. Lin28A is associated with fibroblast and sarcoma cell reprogramming, whereas its homologue Lin28B is associated with hematopoietic cell reprogramming. This study aimed to investigate the expression and prognostic difference between Lin28A and Lin28B in oral squamous cell carcinoma (OSCC). Expression level was assessed by immunohistochemistry and staining location was confirmed by immunofluorescence. Prognostic values were analysed and compared by the Kaplan–Meier analysis and uni and multivariate Cox regression models. Besides, in vitro cell assays and in vivo nude mice xenograft were used to demonstrate the influence of increased Lin28B expression in OSCC. Lin28A and Lin28B expression increased in OSCC, and co-expression of Lin28A and Lin28B showed no significant association with patient prognosis. Kaplan–Meier analysis showed that patients with high Lin28B but not Lin28A expression had lower overall survival (OS) rates than those with low Lin28B expression. Further Univariate analysis showed that patients with increased Lin28B expression had shorter disease-free survival (DFS) and shorter OS, while multivariate analysis showed Lin28B overexpression with TNM stage predicted poor prognosis in patients with OSCC. Besides, stable expressing Lin28B in oral cancer cells promoted cell migration, invasion, colony formation, in vivo proliferation and increased the expression of cancer suppressor miRNA let-7 targeted genes IL-6, HMGA2, the EMT markers Snail and Twist, the angiogenesis inducer VEGF, and the apoptosis inhibitor Survivin. These combined results indicate that Lin28B is a novel marker for predicting prognosis in patients with OSCC and may be a therapeutic target.

Increased expression of peroxiredoxin 6 and cyclophilin A in squamous cell carcinoma of the tongue

OBJECTIVES The aim of this study was to assess the expression levels of two proteins, such as PRDX6 and cyclophilin A (CypA), and to evaluate their relationship with clinicopathologic features and survival in tongue squamous cell carcinomas (TSCCs). MATERIAL AND METHODS An immunohistochemical study was performed comprising a total of 42 tissue samples of patients suffering from TSCCs as well as 10 corresponding adjacent normal tissues. After detection of PRDX6 and CypA, their expression levels were semiquantitatively evaluated and correlated with clinicopathologic variables. RESULTS   Both PRDX6 and CypA expressions were significantly higher in tissue samples of TSCCs compared with the 10 corresponding adjacent normal tissues (P < 0.01). A statistically significant correlation in TSCCs regarding the expression of PRDX6 and CypA was revealed (P = 0.005), and the lymphadenectasis was correlated with PRDX6 (P < 0.05). Results of a multivariate analysis revealed age, CypA expression, cervical lymph node metastases, and tongue cancer differentiation to be independent prognostic variables in respect of the overall survival rate (P < 0.05). CONCLUSIONS It could be detected that PRDX6 and CypA are associated with tumorigenesis in TSCCs. High levels of CypA expression may predict reduced survival time.

NOTCH1 inhibition enhances the efficacy of conventional chemotherapeutic agents by targeting head neck cancer stem cell

Cancer stem cells (CSCs) are considered responsible for tumor initiation and chemoresistance. This study was aimed to investigate the possibility of targeting head neck squamous cell carcinoma (HNSCC) by NOTCH1 pathway inhibition and explore the synergistic effect of combining NOTCH inhibition with conventional chemotherapy. NOTCH1/HES1 elevation was found in human HNSCC, especially in tissue post chemotherapy and lymph node metastasis, which is correlated with CSCs markers. NOTCH1 inhibitor DAPT (GSI-IX) significantly reduces CSCs population and tumor self-renewal ability in vitro and in vivo. Flow cytometry analysis showed that NOTCH1 inhibition reduces CSCs frequency either alone or in combination with chemotherapeutic agents, namely, cisplatin, docetaxel, and 5-fluorouracil. The combined strategy of NOTCH1 blockade and chemotherapy synergistically attenuated chemotherapy-enriched CSC population, promising a potential therapeutic exploitation in future clinical trial.

Overexpression of thioredoxin system proteins predicts poor prognosis in patients with squamous cell carcinoma of the tongue.

The human thioredoxin (Trx) system plays a critical role in the regulation of cellular reduction-oxidation (redox) homeostasis, which has been widely investigated in several types of cancer because of its association with cell growth and anti-apoptosis progress. This study aimed to evaluate the expression of Trx and Trx reductase-1 (TrxR-1) and explore the potential role of these proteins in tongue squamous cell carcinoma (TSCC). Immunohistochemistry was employed to analyze the protein expression levels of Trx and TrxR-1 in 65 TSCC tissue samples and 10 normal oral mucosa samples. The results were then evaluated semiquantitatively and compared to other clinicopathological variables. Both Trx and TrxR-1 expression levels were significantly higher in TSCC tissues as compared with the 10 normal oral mucous samples (P<0.01). A highly significant association between Trx and TrxR-1 expression in TSCCs was revealed (P=0.001), and the expression of Trx was correlated with tumour cell differentiation (P=0.001). Moreover, Kaplan-Meier analysis revealed that Trx expression and TNM stage were significantly related with 5-year survival rate (P=0.033, 0.000), while TrxR-1 expression was not associated with survival (P=0.092). The results indicated that high expression of Trx and TrxR-1 was associated with tumourigenesis in TSCC, and overexpression of Trx might predict poor prognosis.