Hypoxia inducible factor‐1α expression in areca quid chewing‐associated oral squamous cell carcinomas

Oral Diseases (2010) 16 , 696–701 Objectives: Hypoxia inducible factor (HIF)‐1α gene expression is mainly induced by tissue hypoxia. Overexpression of HIF‐1α has been demonstrated in a variety of cancers. The aim of this study was to compare HIF‐1α expression in normal human oral epithelium and areca quid chewing‐associated oral squamous cell carcinoma (OSCC) and further to explore the potential mechanisms that may lead to induce HIF‐1α expression. Methods: Twenty‐five OSCC from areca quid chewing‐associated OSCC and 10 normal oral tissue biopsy samples without areca quid chewing were analyzed by immunohistochemistry. The oral epithelial cell line GNM cells were challenged with arecoline, a major areca nut alkaloid, by using Western blot analysis. Furthermore, glutathione precursor N‐acetyl‐l ‐cysteine (NAC), AP‐1 inhibitor curcumin, extracellular signal‐regulated protein kinase inhibitor PD98059, and protein kinase C inhibitor staurosporine were added to find the possible regulatory mechanisms. Results: Hypoxia inducible factor‐1α expression was significantly higher in OSCC specimens than normal specimen (P < 0.05). Arecoline was found to elevate HIF‐1α expression in a dose‐ and time‐dependent manner (P < 0.05). The addition of NAC, curcumin, PD98059, and staurosporine markedly inhibited the arecoline‐induced HIF‐1α expression (P < 0.05). Conclusions: Hypoxia inducible factor‐1α expression is significantly upregulated in areca quid chewing‐associated OSCC and HIF‐1α expression induced by arecoline is downregulated by NAC, curcumin, PD98059, and staurosporine.     

Phospholipase C‐γ1 expression correlated with cancer progression of potentially malignant oral lesions

Background: Phospholipase C‐γ1 (PLCγ1) is required for cellular migration during tumor progression and invasion of oral squamous cell carcinoma (OSCC) cells. The objective of the current study was to determine immunoexpression pattern of PLCγ1 in oral potentially malignant lesions (OPLs) and evaluate PLCγ1 usefulness as a biomarker for predicting clinical behavior in the carcinogenesis of OPL. Methods: In a retrospective follow‐up study, the expression pattern of PLCγ1 protein was determined using immunohistochemistry in samples from 68 patients, including untransformed cases (n = 38) and malignant‐transformed cases (n = 30). The corresponding post‐malignant lesions (OSCCs) were also performed. Results: We observed that elevated expression of PLCγ1 in 40 of 68 (59%) general OPLs and 23 of 30 (77%) OSCCs compared with that in normal oral mucosa. Kaplan–Meier analysis revealed that patients with PLCγ1 positivity had a significantly higher incidence of OSCC than those with PLCγ1 negativity. Cox regression analysis revealed that PLCγ1 expression patterns were significantly associated with increased risk of malignant progression. In addition, the correlation between PLCγ1 expression in pre‐malignant OPL and that in post‐malignant OSCC was significant (P = 0.004). Conclusion: These data indicate that PLCγ1 expression in OPL correlated with oral cancer progression, and PLCγ1 may serve as a useful marker for the identification of high‐risk OPL into OSCC.     

Histone deacetylase‐1 and ‐2 expression in mobile tongue squamous cell carcinoma: associations with clinicopathological parameters and patients survival

J Oral Pathol Med (2011) 40 : 706–714 Background: Histone deacetylases (HDACs) have been associated with tumor development and progression in several types of human malignancy and HDAC inhibitors are currently being explored as anti‐cancer agents in clinical trials. The aim of the present study was to evaluate the clinical significance of HDAC‐1 and ‐2 protein expression in mobile tongue squamous cell carcinoma (SCC). Methods: HDAC‐1 and ‐2 protein expression was assessed immunohistochemically on 49 mobile tongue SCC tissue samples and was analyzed in relation with clinicopathological characteristics, overall and disease‐free patients’ survival. Results: HDAC‐1 overexpression was significantly associated with younger patients’ age (P = 0.0381) and male gender (P = 0.0345), poor histopathological grade of differentiation (P = 0.0236) and the presence of lymph node metastases (P = 0.0104). Intense HDAC‐1 staining intensity was significantly associated with male gender (P = 0.0127), increased stromal infiltration reaction (P = 0.0125) and well‐defined shape of tumor invasion (P = 0.0396). HDAC‐2 overexpression did not show significant correlations with any clinicopathological parameters, whereas intense HDAC‐2 staining intensity was significantly associated with the presence of muscular invasion (P = 0.0466) and advanced depth of invasion (P = 0.0251). Mobile tongue SCC patients with HDAC‐1 overexpression presented shorter overall and disease‐free survival compared to those with no evidence of HDAC‐1 overexpression (log‐rank test, P = 0.0651 and 0.0247, respectively). Conclusions: The present study supported evidence that HDACs may participate in the formation and progression of mobile tongue SCC, reinforcing their possible use as biomarkers as also the therapeutic utility of HDAC inhibitors in mobile tongue SCC chemoprevention and treatment.     

Insulin‐like growth factor II mRNA‐binding protein 3 expression promotes tumor formation and invasion and predicts poor prognosis in oral squamous cell carcinoma

J Oral Pathol Med (2011) 40 : 699–705 Background: Insulin‐like growth factor II mRNA‐binding protein 3 (IGF2BP3), an oncofetal RNA‐binding protein, has been implicated in the enhancement of proliferation and invasion in various cancers. This study aimed to investigate the clinical significance and functional role of IGF2BP3 expression in oral squamous cell carcinoma (OSCC). Methods: IGF2BP3 expression in 93 OSCC patients was investigated using immunohistochemical staining and correlated with clinical parameters and patients’ survival. The effect of IGF2BP3 on cell invasion ability was evaluated by RNA interference in OSCC cell line. Results: High expression of IGF2BP3 in OSCC was significantly correlated with large tumor size and lymph node metastasis. Kaplan‐Meier analysis revealed that oral cancer patients with high IGF2BP3 expression had a significantly lower 5‐year survival (P = 0.0017). Multivariate analysis of clinical samples demonstrated IGF2BP3 to be an independent prognosis factor (P = 0.003). Moreover, the IGF2BP3 shRNA significantly suppressed the invasion ability of OSCC in vitro, and the knockdown of endogenous IGF2BP3 expression also inhibited tumor formation in vivo. Conclusions: IGF2BP3 enhances cell invasion ability and tumorigenicity in human OSCC in vitro and in vivo. IGF2BP3 is an independent prognostic factor in patients with OSCC. Targeting of IGF2BP3 could potentially suppress the tumor growth and metastasis to improve the outcome of patients with OSCC.     

Co‐expression of Hif1α and CAIX is associated with poor prognosis in oral squamous cell carcinoma patients

J Oral Pathol Med (2010) 39 : 313–317 Background: This study investigates the prognostic impact of the expression of hypoxia‐inducible factor 1α (Hif1α) and carbonic anhydrase IX (CAIX) detected by immunohistochemistry in oral squamous cell carcinoma (OSCC). Methods: Statistical analysis of immunohistochemical results with clinical parameters including survival outcomes was performed for 80 OSCC patients. Results: Patients with a low expression of both proteins survived on average 54.8 months, whereas those with an increased expression of Hif1α in their tumors combined with a low expression of CAIX survived on average only 37.6 months (P = 0.026). In multivariate Cox’s regression hazard analysis, again patients with a low expression of Hif1α/CAIX had the best prognosis, whereas patients with increased Hif1α and low CAIX expression carried a 4.97‐fold increased risk of tumor‐related death (P = 0.042). Conclusion: A co‐detection of low Hif1α/CAIX expression is significantly correlated with a better prognosis for OSCC patients, which may have implications for therapy options for these patients.     

Fos‐related activator‐1 is overexpressed in oral squamous cell carcinoma and associated with tumor lymph node metastasis

J Oral Pathol Med (2010) 39 : 470–476 Background: Previously, we established an in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC), including the human immortalized oral epithelia cells (HIOECs) and its derived cancerous HB cells. Then, expression microarray analysis showed that the gene encoding fos‐related activator‐1 (Fra‐1) was significantly upregulated in the cancerous HB cells compared with HIOECs. Methods: To confirm the expression of Fra‐1 at mRNA and protein levels by real‐time PCR and western blotting analysis in the carcinogenesis model of OSCC and CAL27 cells. To investigate Fra‐1 expression in clinical samples from 30 primary OSCC patients by immunohistochemistry. Results: Fra‐1 expression was increased both at mRNA and protein levels in this carcinogenesis model of OSCC and CAL27 cells. Nuclear and cytoplasmic Fra‐1 protein expressions both increased in the cancerous tissues compared with those in the paired adjacent non‐malignant epithelia (nuclear: P < 0.001, cytoplasmic: P = 0.003). A higher level of nuclear Fra‐1 expression was seen in the tumor samples of patients with lymph node metastasis than those without lymph node metastasis (5.07 ± 1.33 vs 3.81 ± 1.33, P = 0.023). Higher level of Fra‐1 expression was also found in the tumor invasive margin than tumor center. Conclusions: Fra‐1 is a positive gene of OSCC development and progression, Fra‐1 can be used as a potential therapeutic target gene and an additional marker for evaluation of lymph node metastasis.     

Effect of silencing of mediator of DNA damage checkpoint protein 1 on the growth of oral squamous cell carcinoma in vitro and in vivo

Mediator of DNA damage checkpoint protein 1 (MDC1) is involved in DNA damage repair and has been linked to tumor invasion, metastasis, and prognosis. This study investigated the effects of MDC1 in oral squamous cell carcinoma (OSCC) in vitro and in vivo. RNA interference‐mediated knockdown of MDC1 was performed in two OSCC cell lines (Tca‐8113 and KB). Real‐time PCR and western blotting were performed to determine expression of mRNA and protein, respectively, of MDC1. Cell viability was assessed using the MTT assay. Colony‐formation assays were performed by staining with 0.5% crystal violet. Cell migration and invasion were detected by Transwell assays. The role of MDC1 in OSCC was examined in vivo via injection of Tca‐8113 cells transfected with MDC1 small interfering (si)RNA or negative‐control siRNA into a mouse xenograft model of OSCC. Our results showed that MDC1 knockdown decreased cell proliferation. Inhibition of MDC1 decreased colony formation of Tca‐8113 and KB cells by 62% and 68%, respectively, and MDC1 knockdown reduced the number of migratory and invasive cells compared with the control group. Moreover, the xenograft mouse model of MDC1 knockdown showed reduced tumor growth. Our study suggests that MDC1 plays a role in tumorigenesis and might be a potential target for the treatment of patients with OSCC.     

Cancer-associated fibroblasts and CD163-positive macrophages in oral squamous cell carcinoma: their clinicopathological and prognostic significance

J Oral Pathol Med (2012) 41 : 444–451 Background: Stromal cells are believed to affect cancer invasion and metastasis. The purpose of this study was to evaluate the distribution of cancer‐associated fibroblasts (CAFs) and the incidence of tumor‐associated macrophages (TAMs) in oral squamous cell carcinoma (OSCC), focusing on clinicopathological factors and patient prognosis, as well as cancer invasion. Methods: The study included 108 patients with OSCC. Anti‐α‐smooth muscle actin, CD68, and CD163 antibodies were used to identify CAFs and TAMs. CAFs were divided into 4 grades on the basis of staining intensity: negative (0), scanty (1), focal (2), and abundant (3). The most intensive areas of macrophage concentration in each tumor invasive stroma were also evaluated. Results: The cancer specimens were divided into Grade 0/1, Grade 2, and Grade 3 on the basis of CAF grade. In addition, they were divided into low‐ and high‐grade groups on the basis of the number of CD68‐positive and CD163‐positive macrophages. The latter were significantly increased in the Grade 2 CAF group compared to the Grade 0/1 group (P = 0.009). Kaplan–Meier and multivariate survival analyses revealed that Grade 2 CAFs (P = 0.003) and high CD163‐positive macrophage levels (P = 0.007) significantly correlated with a poor outcome in patients with OSCC, and that a high CD163‐positive macrophage level was a significant and an independent prognostic factor (P = 0.045). Conclusions: Cancer‐associated fibroblasts and CD163‐positive macrophages may be potential prognostic predictors of OSCC.     

POLD1对口腔鳞癌细胞增殖、侵袭的影响及调控机制

目的:探讨人源DNA聚合酶 δ 催化亚基(POLD1)的表达对口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)细胞增殖、迁移、侵袭、细胞周期的影响及其相关机制.方法:使用生物信息数据分析POLD1在头颈部鳞癌及癌旁组织中的表达,利用免疫组织化学染色检测POLD1在40对口腔鳞癌及癌旁...     

口腔鳞癌中HIF-1α和CD44v6的表达及其相关性研究

目的：探讨CD44v6、HIF-1α在口腔鳞状细胞癌（OSCC）中的表达、两者相关性及其作用。方法：采用免疫组织化学方法检测HIF-1α和CD44v6在65例OSCC和20例正常非肿瘤组织中的表达情况、以及两者的相关性。结果：HIF-1α和CD44v6在OSCC中的阳性表达率分别为83.08%和87.69%,均高于正常组织15.0%和10.0%;差异均有显著性意义（P〈0.01）;CD44v6和HIF-1α在OSCC中的表达呈正相关关系（r=0.391,P〈0.01）。结论：HIF-1α和CD44v6在OSCC组织中呈过表达,二者之间的相互作用与OSCC增殖、侵袭和转移密切相关。     

Markedly increased Oct4 and Nanog expression correlates with cisplatin resistance in oral squamous cell carcinoma

J Oral Pathol Med (2011) 40 : 621–628 Background: Oral squamous cell carcinoma (OSCC) is the sixth most prevalent cancer worldwide. Cancer stem cells (CSC) model theoretically contribute to tumor growth, metastasis, and chemo‐radioresistance. Cisplatin is a widely used chemotherapeutic agent for OSCC treatment. The aim of this study was to compare stemness genes expression in chemo‐sensitive and chemo‐resistant specimens and further explore the potential markers that may lead to induce chemo‐resistance in OSCC. Methods: The study method is the treatment of OC2 cells with cisplatin select cisplatin‐resistant OC2 cells. Self‐renewal ability was evaluated by cultivating parental and cisplatin‐resistant OC2 cells within sphere‐forming assay after serial passages. Differential expression profile of stemness markers between parental and cisplatin‐resistant OC2 cells was elucidated. The parental and cisplatin‐resistant OC2 cells were assessed for migration/invasion/clonogenicity tumorigenic properties in vitro. Expression of stemness markers in chemo‐sensitive and chemo‐resistant patients with OSCC was performed by immunohistochemistry staining in vivo. Results: Sphere‐forming/self‐renewal capability was increased in cisplatin‐resistant OC2 cells. Cisplatin‐resistant OC2 cells highly expressed the stemness markers (Nanog, Oct4, Bmi1, CD117, CD133, and ABCG2). Furthermore, cisplatin‐resistant OC2 cells increased migration/invasion/clonogenicity ability. Notably, up‐regulation of Oct4 and Nanog expression was significantly observed in cisplatin‐resistant patients with OSCC (**P < 0.01). Conclusions: These data indicate that cancer stem‐like properties were expanded during the acquisition of cisplatin resistance in OSCC. Clinically, oral cancer stemness markers (Oct4 and Nanog) overexpression may promote the OSCC’s recurrence to resist cisplatin.