BCL10 expression is unrelated to clinico-pathological parameters or prognoses for oral squamous cell carcinomas

Abstract

BCL-10 (B-cell lymphoma 10) has been linked to a pro-apoptotic gene in mucosa-associated lymphoid tissue (MALT) lymphomas. We describe the expression of BCL10 in oral squamous cell carcinoma (OSCC) and its relation to clinical, pathological and prognostic parameters. We carried out a retrospective study of 50 patients in Spain who were diagnosed with OSCC. We constructed a tissue microarray of the samples to study the expression of BCL10 using immunohistochemistry. Diffuse and homogeneous staining was observed in the nuclei and cytoplasms of most neoplastic cells of the vast majority of tumors and no significant differences were seen in different areas of the tumors. The expression was unrelated to any clinical or pathological parameters including tumor stage. The intra-class coefficient was 0.97, which indicates the minimal variability among the determinations.     

The use of tissue microarrays for semiquantitative evaluation of ATPaseC1 expression is ineffective

Abstract

We described earlier the possible role of ATPaseC1 expression as a diagnostic and prognostic marker for oral cancer; others have reported its use for tumors of the lung and breast. We assessed ATPaseC1 expression in a sample of oral squamous cell carcinoma (OSCC) using tissue microarrays (TMAs) to analyze the relation between ATPaseC1 expression and clinical, histopathological and prognostic parameters. We performed a retrospective study of 48 cases of OSCC. We constructed TMAs using two different regions of each tumor. V-ATPaseC1 immunohistochemistry was performed and assessed semiquantitatively. ATPaseC1 staining was observed in most of the neoplastic cells in all tumors. Staining was diffusely cytoplasmic and, to a lesser extent, nuclear. The degree of concordance between the measurements performed in tissue microarray 1 (TMA1) and tissue microarray 2 (TMA2), as evaluated using the intra-class correlation coefficient (ICC), was low. We found great variability in the immunohistochemical staining of the different regions of each tumor. We found 16 cases with mild expression (33.3%), 20 with moderate expression (41.7%) and 12 with intense expression (25%). Differences in the clinical-pathological variables studied were not statistically significant. The difficulty of immunohistochemical evaluation, the heterogeneity of the carcinomas and the fact that evaluation of expression requires semiquantitative analysis render the reliability of the results obtained from TMA-based techniques questionable.     

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.     

DAPK1、hTERT在口腔鳞癌中的表达及其临床意义研究

目的:探讨凋亡相关蛋白激酶1(DAPK1)、端粒酶催化亚单位(hTERT)在口腔鳞状细胞癌(OSCC)中的表达,并探讨其临床意义。方法:应用免疫组织化学方法检测DAPK1、hTERT在93例OSCC组织及10例癌旁正常组织中的表达,并分析其与OSCC临床病理参数之间的关系及其在OSCC浸润前沿中的作用。结果:癌旁正常组织中DAPK1的表达显著高于OSCC组织中DAPK1的表达,且高、中、低分化OSCC组织中DAPK1的表达比较均有显著差异(P0.05),OSCC浸润前沿组织中DAPK1的表达低于非前沿部分(P0.05),而DAPK1的表达与OSCC浸润前沿的IFG总分无显著相关性(P0.05)。癌旁正常组织中hTERT的表达显著低于OSCC组织中hTERT的表达(P0.05),且高、中、低分化OSCC组织中hTERT的表达比较均有显著差异(P0.05);OSCC浸润前沿组织中hTERT的表达高于非前沿部分(P0.05),且与OSCC浸润前沿的IFG总分有关(P0.05)。DAPK1、hTERT的表达均与OSCC患者的性别、年龄、淋巴结转移无显著相关性(P0.05)。结论:口腔鳞状细胞癌组织,特别是其前沿组织中DAPK1的表达显著下调和hTERT的表达明显上调,可能通过阻碍口腔鳞状细胞癌细胞凋亡,共同促进口腔鳞状细胞癌的生长、分化和浸润。     

环氧化酶-2在口腔鳞癌中的表达及其意义

目的探讨环氧化酶-2(COX-2)的表达与口腔鳞癌生物学行为的关系及其意义.方法采用免疫组织化学S-P法检测10例正常口腔黏膜、12例炎症组织和62例口腔鳞癌中COX-2的表达,并结合临床病理资料进行分析.结果口腔鳞癌中COX-2的表达明显高于正常口腔黏膜和炎症组织(P<0.001).COX-2的表达与口腔鳞癌的部位、大小、临床分期及淋巴结转移无明显相关,但与病理分级相关性显著,随分化程度的降低而增强(P=0.002).结论 COX-2很可能在口腔鳞癌的发生、发展中扮演重要角色,抑制COX-2的活性有望成为口腔鳞癌防治的新途径.     

Role of EZH2 in oral squamous cell carcinoma carcinogenesis

Oral squamous cell carcinoma (OSCC) is a common human malignancy with high incidence rate and poor prognosis. Although the polycomb group protein enhancer of zeste homolog 2 (EZH2) plays a crucial role in cell proliferation and differentiation during the occurrence and development progress of several kinds of malignant tumors, the impact of EZH2 on the development and progression of OSCC is unclear. In this study, we demonstrate that EZH2 is overexpressed in OSCC cells and clinical tissue. With in vitro RNAi analysis, we generated stable EZH2 knocking down cell lines from two OSCC cell lines, with two sh-RNAs targeting to EZH2, respectively. We found that knocking down of EZH2 could decrease the proliferation ability and induce apoptosis of OSCC cells. Moreover, we demonstrated that of EZH2 inhibition decreased the migration and metastasis of OSCC cells. In conclusion, the results of the current study demonstrated an association between EZH2 expression and OSCC cell development. We recommend that EZH2 acts as an oncogene and plays an important role in OSCC carcinogenesis.     

Differential Impact of IL-10 Expression on Survival and Relapse between HPV16-Positive and -Negative Oral Squamous Cell Carcinomas

Human papillomavirus (HPV) is a risk factor in a subset of oropharyngeal cancer; however, the contribution of HPV in the malignancy of oral squamous cell carcinomas (OSCC) is not fully understood in Taiwanese. Herein, 61 patients with no risk factors and 117 patients with one or more risk factors were enrolled in this study. HPV16/18 infection rate in non-smokers, non-drinkers and non-betel quid chewers was higher than their counterparts. The development of HPV-infected cancer has been shown to be associated with interleukin-10 (IL-10) expression. To this end, IL-10 mRNA expression in OSCC tumors was evaluated by real-time RT-PCR. Data showed that HPV-positive patients had higher IL-10 mRNA levels than in HPV-negative patients. Kaplan-Meier and Cox-regression analysis indicated that the prognostic significance of IL-10 mRNA on overall survival and relapse free survival was only observed in HPV-positive OSCC, but not in HPV-negative OSCC. Mechanistically, the elevation of IL-10 by E6 was responsible for increased colony formation and migration capability in OSCC cells. Therefore, we suggest that IL-10 induced by E6 promotes cell growth and migration capability and consequent poor survival and relapse in HPV-positive OSCC.     

A study on the differences between oral squamous cell carcinomas and normal oral mucosas measured by Fourier transform infrared spectroscopy

We investigated the differences of Fourier transform infrared (FTIR) spectra between oral squamous cell carcinoma (OSCC) and normal gingival epithelium (NGE) or normal subgingival tissue (NST). We used 15 specimens of OSCC which had not been treated before measurement and 10 of NGE or NST. We also used cultured oral squamous cell carcinoma (COSCC) and the tissue (MSCC) which massed for 3 months after the cultured oral squamous cell carcinoma was transplanted into the lower back of a rat. Those tissue spectra were compared with the purified human collagens and human keratin. One half of every tissue specimen was measured with FTIR and the other half was investigated histologically. The differences of FTIR spectra between OSCC and NGE were observed in the bands between 1431 and 1482 cm(-1) and between 1183 and 1274 cm(-1). The shoulder at 1368 cm(-1) tended to disappear in OSCC, and the peaks at 1246 and 1083 cm(-1) found in NGE tended to shift to those at 1242 and 1086 cm(-1) in OSCC, respectively. The infrared spectrum of NST was noticed to be strongly influenced by the presence of collagen. Significant differences were also observed in the second derivative FTIR spectra between OSCC and NGE. Our data suggested that this infrared technique is applicable to clinical diagnostics.     

miR-21 inhibitor sensitizes human OSCC cells to cisplatin

miR-21 as a tumor oncogenic molecule has been reported. However, whether miR-21 can affect the sensitivity of oral squamous cell carcinoma (OSCC) cells to cisplatin remain unclear. The aim of this study is to evaluate the roles of miR-21 in the sensitivity of OSCC cells to cisplatin. RT-PCR assay was performed to detect the expression of miR-21 in 10 pairs of OSCC and noncancerous tissue samples. Then As-miR-21 oligonucleotides were used to down the miR-21 expression. Finally, the effects of miR-21 downregulation the sensitivity of OSCC cells (CA-27) to cisplatin in vitro were also detected. The level of miR-21 expression in OSCC tissues was significantly higher than that in corresponding noncancerous tissues. Down the expression of miR-21 could significantly inhibit growth and induce apoptosis of CA-27 cells. Moreover, downregulation of miR-21 could sensitize CA-27 cells to cisplatin possibly by increasing cisplatin induced apoptosis. This study demonstrated that combination of cisplatin application with miR-21 downregulation might be a potential strategy for the treatment of human OSCC.     

Ubiquitin D Promotes Progression of Oral Squamous Cell Carcinoma via NF-Kappa B Signaling

Ubiquitin D (UBD) is highly upregulated in many cancers, and plays a pivotal role in the pathophysiological processes of cancers. However, its roles and underlying mechanisms in oral squamous cell carcinoma (OSCC) are still unclear. In the present study, we investigated the role of UBD in patients with OSCC. Quantitative real-time polymerase chain reaction and Western blot were used to measure the expression of UBD in OSCC tissues. Immunohistochemistry assay was used to detect the differential expressions of UBD in 244 OSCC patients and 32 cases of normal oral mucosae. In addition, CCK-8, colony formation, wound healing and Transwell assays were performed to evaluate the effect of UBD on the cell proliferation, migration, and invasion in OSCC. Furthermore, a xenograft tumor model was established to verify the role of UBD on tumor formation in vivo. We found that UBD was upregulated in human OSCC tissues and cell lines and was associated with clinical and pathological features of patients. Moreover, the overexpression of UBD promoted the proliferation, migration and invasion of OSCC cells; however, the knockdown of UBD exerted the opposite effects. In this study, our results also suggested that UBD promoted OSCC progression through NF-κB signaling. Our findings indicated that UBD played a critical role in OSCC and may serve as a prognostic biomarker and potential therapeutic target for OSCC treatment.     

Downregulation of FAP suppresses cell proliferation and metastasis through PTEN/PI3K/AKT and Ras-ERK signaling in oral squamous cell carcinoma

It is largely recognized that fibroblast activation protein (FAP) is expressed in cancer-associated fibroblasts (CAFs) of many human carcinomas. Furthermore, FAP was recently also reported to be expressed in carcinoma cells of the breast, stomach, pancreatic ductal adenocarcinoma, colorectum, and uterine cervix. The carcinoma cell expression pattern of FAP has been described in several types of cancers, but the role of FAP in oral squamous cell carcinoma (OSCC) is unknown. The role of endogenous FAP in epithelium-derived tumors and molecular mechanisms has also not been reported. In this study, FAP was found to be expressed in carcinoma cells of OSCC and was upregulated in OSCC tissue samples compared with benign tissue samples using immunohistochemistry. In addition, its expression level was closely correlated with overall survival of patients with OSCC. Silencing FAP inhibited the growth and metastasis of OSCC cells in vitro and in vivo. Mechanistically, knockdown of FAP inactivated PTEN/PI3K/AKT and Ras-ERK and its downstream signaling regulating proliferation, migration, and invasion in OSCC cells, as the inhibitory effects of FAP on the proliferation and metastasis could be rescued by PTEN silencing. Our study suggests that FAP acts as an oncogene and may be a potential therapeutic target for patients with OSCC.     

Valosin-Containing Protein (VCP/p97)-Expression Correlates with Prognosis of HPV- Negative Oropharyngeal Squamous Cell Carcinoma (OSCC)

Valosin-containing protein (VCP)/p97 has been shown to be associated with antiapoptotic function via activation of the nuclear factor-B (NFB) signaling pathway and with metastasizing of tumors in several studies. VCP is located on chromosome 9p13-p12, a region often deleted in oropharyngeal squamous cell carcinoma (OSCC). The clinical significance of VCP expression in OSCC however remains unclear. In this study, expression of VCP was determined in 106 patients (77 male (71.3%) and 31 female (28.7%); age-range: 34–79 years (mean age 57 years)) by immunohistochemistry and in a subset of 15 patients by quantitative PCR. HPV-DNA was detected by polymerase chain reaction and p16^INK4a immunohistochemistry. The experimental findings were correlated with clinico-pathological data and survival parameters. 47.2% of all OSCC specimens were analyzed as negative or weak staining intensity for VCP. 52.8% of all specimens showed a high staining intensity for VCP. 73.1% of all patients were tested HPV-negative, 26.9% were HPV-positive. The 5-year disease-free and overall survival probabilities of all patients were 71.2% and 55.7%, respectively. No correlation could be found between HPV-status and VCP expression. VCP overexpression in HPV-negative patients was associated with significantly better 5-year disease-free survival (86.4% vs., 45.6%, p = 0.017). The level of VCP-intensity determined by immunohistochemistry could be an additional prognostic marker in HPV-negative OSCC. VCP expression seems not to correlate with the HPV-status.     

Topotecan and methotrexate alter expression of the apoptosis-related genes BCL2, FAS and BCL2L12 in leukemic HL-60 cells

The BCL2 family of genes (B-cell CLL/lymphoma 2; Bcl-2) plays a pivotal role in the highly regulated process of apoptosis. We have recently cloned a newly identified member of this family, BCL2L12, which was found to be differentially expressed in many tumors. It is known that topotecan and methotrexate act through induction of apoptosis in cancer cells. In the present study we investigated the expression profile of the novel apoptotic gene BCL2L12 in relation to other apoptotic genes in the human leukemic cell line HL-60, after treatment with topotecan or methotrexate. The kinetics of apoptosis induction and cell toxicity were investigated by DNA laddering and the MTT method, respectively. Gene expression levels were analyzed by RT-PCR using gene-specific primers. Downregulation of BCL2L12, BCL2 and FAS was observed after treatment of HL-60 cells with topotecan, while treatment with methotrexate led to downregulation of BCL2 and FAS, with no change in BCL2L12 expression. Our results support the significance of mRNA modulations in the expression of apoptosis-related genes during treatment of human leukemic cells with anticancer drugs.     

Ubiquitin-specific protease 14 promotes radio-resistance and suppresses autophagy in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is a common malignant tumor in the world. Radiotherapy is one of the standard therapies for patients with OSCC, but its clinical efficiency is limited due to radioresistance. In this study, we identified a mechanism of such resistance regulated by Ubiquitin-specific protease 14 (USP14). USP14 expression was significantly increased in clinical OSCC tissue samples and cell lines, and OSCC patients with high USP14 expression predicted poor overall survival rate. Additionally, a negative correlation between USP14 and LC3B was observed in patients with OSCC. We then found that irradiation (IR)-reduced cell survival of OSCC cells lines was further decreased when USP14 was knocked down. However, USP14 over-expression significantly promoted the cell viability of OSCC cells after IR treatment. Colony formation analysis confirmed thatafter IR treatment,USP14 knockdown markedly decreased the proliferation of OSCC cells, but over-expressing USP14 significantly up-regulated the proliferative activity of OSCC cells. Furthermore, DNA damage caused by IR was enhanced by USP14 knockdown, while been suppressed in OSCC cells with USP14 over-expression. Additionally, IR-inducedapoptosis was further promoted by USP14 knockdown in OSCC cells, which was, however, significantly abolished by USP14 over-expression.Moreover, our in vivo studies showed that IR-reduced tumor growth and tumor weight were further enhanced by USP14 knockdown in OSCC tumor-bearing nude mice. Finally, we found that USP14 knockdown could promote IR-induced autophagy by increasing LC3BII and γH2AX expression levels in IR-treated OSCC cells. However, this event was markedly abolished by ATG5 knockdown, subsequently restoring the cell proliferation in IR-incubated OSCC cells.Finally, we found that USP14-mediated apoptosis was autophagy-dependent in IR-treated OSCC cells. Taken together, these findings suggested that suppressing USP14 could alleviateradioresistancein OSCC both in vitro and in vivo by inducing apoptosis and autophagy, and thus could be served as a promising therapeutic strategy for OSCC treatment.     

Fibroblast Growth Factor (FGF-2) and Its Receptors FGFR-2 and FGFR-3 May Be Putative Biomarkers of Malignant Transformation of Potentially Malignant Oral Lesions into Oral Squamous Cell Carcinoma

There are several factors like angiogenesis, lymphangiogenesis, genetic alterations, mutational factors that are involved in malignant transformation of potentially malignant oral lesions (PMOLs) to oral squamous cell carcinoma (OSCC). Fibroblast growth factor-2 (FGF-2) is one of the prototypes of the large family of growth factors that bind heparin. FGF-2 induces angiogenesis and its receptors may play a role in synthesis of collagen. FGFs are involved in transmission of signals between the epithelium and connective tissue, and influence growth and differentiation of a wide variety of tissue including epithelia. The present study was undertaken to analyze expression of FGF-2 and its receptors FGFR-2 and FGFR-3 in 72 PMOLs, 108 OSCC and 52 healthy controls, and their role in risk assessment for malignant transformation of Leukoplakia (LKP) and Oral submucous fibrosis (OSMF) to OSCC. Immunohistochemistry was performed using antibodies against FGF-2, FGFR-2 and FGFR-3. IHC results were validated by Real Time PCR. Expression of FGF-2, FGFR-2 and FGFR-3 was upregulated from PMOLs to OSCC. While 90% (9/10) of PMOLs which showed malignant transformation (transformed) expressed FGF-2, only 24.19% cases (15/62) of PMOLs which were not transformed (untransformed) to OSCC expressed FGF-2. Similarly, FGFR-2 expression was seen in 16/62 (25.81%) of untransformed PMOLs and 8/10 (80%) cases of transformed PMOLs. FGFR-3 expression was observed in 23/62 (37.10%) cases of untransformed PMOLs and 6/10 (60%) cases of transformed PMOLs. A significant association of FGF-2 and FGFR-2 expression with malignant transformation from PMOLs to OSCC was observed both at phenotypic and molecular level. The results suggest that FGF-2 and FGFR-2 may be useful as biomarkers of malignant transformation in patients with OSMF and LKP.     

Urinary-type plasminogen activator receptor (uPAR) modulates oral cancer cell behavior with alteration in p130cas

Oral cavity cancer is among the most frequently diagnosed cancers worldwide and urinary-type plasminogen activator receptor (uPAR) is clinically associated with more invasive tumors and enhanced lymph node metastasis. We seek to further elucidate the mechanism of by which uPAR promotes cell aggressiveness in the unique context of oral squamous cell carcinoma (OSCC). The contribution of uPAR expression to aggressive cellular behavior of OSCC was examined using in vitro cellular models wherein the expression of uPAR was manipulated and in a human OSCC tissue microarray. Results show altered adhesion, motility, and invasion in cells that overexpress uPAR relative to vector control cells. Distinct alterations of focal adhesion protein expression and phosphorylation, including p130cas and paxillin were observed, suggestive of enhanced focal adhesion turnover. Immunohistochemical analysis of microarrayed human OSCC revealed a significant correlation between uPAR and p130cas expression. The non-receptor protein tyrosine kinase c-Src was responsible for the phosphorylation of p130cas in response to uPAR/α3β1/laminin-5 engagement. Further downstream, the Rho family GTPase Cdc42, but not Rac1, was activated, suggesting a pathway leading to actin reorganization, filopodial protrusion and enhanced motility in uPAR overexpressing oral cancer cells. These data shed light on a molecular mechanism whereby acquisition of uPAR expression may modulate OSCC invasive activity through alteration of focal adhesion dynamics.