ThermomiR-377-3p-induced suppression of Cirbp expression is required for effective elimination of cancer cells and cancer stem-like cells by hyperthermia.

BACKGROUND: In recent years, the development of adjunctive therapeutic hyperthermia for cancer therapy has received considerable attention. However, the mechanisms underlying hyperthermia resistance are still poorly understood. In this study, we investigated the roles of cold­inducible RNA binding protein (Cirbp) in regulating hyperthermia resistance and underlying mechanisms in nasopharyngeal carcinoma (NPC). METHODS: CCK-8 assay, colony formation assay, tumor sphere formation assay, qRT-PCR, Western blot were employed to examine the effects of hyperthermia (HT), HT + oridonin(Ori) or HT + radiotherapy (RT) on the proliferation and stemness of NPC cells. RNA sequencing was applied to gain differentially expressed genes upon hyperthermia. Gain-of-function and loss-of-function experiments were used to evaluate the effects of RNAi-mediated Cirbp silencing or Cirbp overexpression on the sensitivity or resistance of NPC cells and cancer stem-like cells to hyperthermia by CCK-8 assay, colony formation assay, tumorsphere formation assay and apoptosis assay, and in subcutaneous xenograft animal model. miRNA transient transfection and luciferase reporter assay were used to demonstrate that Cirbp is a direct target of miR-377-3p. The phosphorylation levels of key members in ATM-Chk2 and ATR-Chk1 pathways were detected by Western blot. RESULTS: Our results firstly revealed that hyperthermia significantly attenuated the stemness of NPC cells, while combination treatment of hyperthermia and oridonin dramatically increased the killing effect on NPC cells and cancer stem cell (CSC)­like population. Moreover, hyperthermia substantially improved the sensitivity of radiation­resistant NPC cells and CSC­like cells to radiotherapy. Hyperthermia noticeably suppressed Cirbp expression in NPC cells and xenograft tumor tissues. Furthermore, Cirbp inhibition remarkably boosted anti­tumor­killing activity of hyperthermia against NPC cells and CSC­like cells, whereas ectopic expression of Cirbp compromised tumor­killing effect of hyperthermia on these cells, indicating that Cirbp overexpression induces hyperthermia resistance. ThermomiR-377-3p improved the sensitivity of NPC cells and CSC­like cells to hyperthermia in vitro by directly suppressing Cirbp expression. More importantly, our results displayed the significantly boosted sensitization of tumor xenografts to hyperthermia by Cirbp silencing in vivo, but ectopic expression of Cirbp almost completely counteracted hyperthermia-mediated tumor cell-killing effect against tumor xenografts in vivo. Mechanistically, Cirbp silencing-induced inhibition of DNA damage repair by inactivating ATM-Chk2 and ATR-Chk1 pathways, decrease in stemness and increase in cell death contributed to hyperthermic sensitization; conversely, Cirbp overexpression-induced promotion of DNA damage repair, increase in stemness and decrease in cell apoptosis contributed to hyperthermia resistance. CONCLUSION: Taken together, these findings reveal a previously unrecognized role for Cirbp in positively regulating hyperthermia resistance and suggest that thermomiR-377-3p and its target gene Cirbp represent promising targets for therapeutic hyperthermia.

High expression of nuclear Snail, but not cytoplasmic staining, predicts poor survival in nasopharyngeal carcinoma.

BACKGROUND: Transcription factor Snail has been shown to promote tumor progression and metastasis in various cancers. However, its clinical significance in nasopharyngeal carcinoma (NPC) is still scanty. We have explored the clinical significance of Snail expression and its association with patient outcome in NPC. METHODS: Immunohistochemistry was used to examine the expression levels of Snail in 122 patients with NPC. RESULTS: Cytoplasmic Snail was detected in 37.7 %, and nuclear staining was detected in 49.2 % of primary tumors, respectively. No significant associations were found between cytoplasmic Snail and the clinicopathologic variables except lymph node metastasis (P = 0.042). However, nuclear Snail was significantly associated with tumor stage (P = 0.003), T classification (P = 0.045), lymph node metastasis (P = 0.019), distant metastasis (P = 0.003), and reduced E-cadherin expression (P = 0.021). Patients with high nuclear Snail expression, but not cytoplasmic staining, had significantly shorter survival than those with low expression (P < 0.001). Significantly, nuclear Snail was an independent prognostic predictor for NPC (P < 0.001). Furthermore, the prognostic impact was largely limited to stage III-IV patients. CONCLUSIONS: We demonstrated first that nuclear Snail, but not cytoplasmic staining, predicts worse outcome. In addition, the prognostic value in stage III-IV suggests that nuclear Snail could be a potential therapeutic target for late stage of NPC patients.

Tumour budding and the expression of cancer stem cell marker aldehyde dehydrogenase 1 in nasopharyngeal carcinoma.

AIMS: To detect the prognostic significance of tumour budding and its expression of aldehyde dehydrogenase 1 (ALDH1) in nasopharyngeal carcinoma (NPC). METHODS AND RESULTS: Tumour budding was investigated in 105 patients with NPC by immunohistochemistry for pan-cytokeratin (AE1/AE3). The intensity of budding correlated strongly with T classification (P=0.008), lymphatic invasion (P<0.001), vascular invasion (P=0.029), lymph node metastasis (P < 0.001), and clinical stage (P=0.010). Univariate analysis revealed that patients with high budding grade had poorer survival than those with low grade (P=0.002). Multivariate analysis showed that tumour budding was an independent predictor of survival (P=0.001). Furthermore, budding cells showed high-level expression of the cancer stem cell (CSC) marker ALDH1. Budding cells with high-level ALDH1 expression contributed to several aggressive behaviours and poor survival (P=0.000). CONCLUSIONS: We describe, for the first time, the presence of tumour budding and its correlation with aggressive tumour behaviour and poor patient survival in NPC. The degree of tumour budding could be a valuable predictive factor in NPC. In addition, we show, also for the first time, that budding cells in NPC might possess the invasive and metastatic properties of CSCs.

Nuclear expression of N-cadherin correlates with poor prognosis of nasopharyngeal carcinoma.

AIMS: To investigate the aberrant expression of N-cadherin in nasopharyngeal carcinoma (NPC) and its prognostic significance. METHODS AND RESULTS: Immunohistochemical staining for N-cadherin protein was performed on tissue microarray (TMA) from 122 NPC patients. Cytoplasmic N-cadherin was observed in 42.6% and nuclear N-cadherin in 45.1% of NPC tissues. High expression of cytoplasmic and nuclear N-cadherin was associated with a majority of the clinicopathological variables, including lymph node metastasis, distant metastasis and clinical stage. Cytoplasmic N-cadherin was associated positively with nuclear N-cadherin expression (P = 0.000). In univariate analysis, cytoplasmic N-cadherin showed no significant impact on patient prognosis. In contrast, the overall survival was significantly shorter in patients with high nuclear N-cadherin than those with low levels of staining (P = 0.002). A high expression of nuclear N-cadherin predicted poorer survival in patients with late stage disease (P = 0.033), but not those with early tumour stage. In addition, multivariate analysis showed nuclear N-cadherin to bean independent prognostic marker for NPC patients (P = 0.024). CONCLUSIONS: Nuclear N-cadherin expression may represent a valuable prognostic marker in NPC patients, especially those with late stage disease.

Neoplastic spindle cells in nasopharyngeal carcinoma show features of epithelial-mesenchymal transition.

AIM: To investigate whether the neoplastic spindle cells in nasopharyngeal carcinoma (NPC) are associated with the process of epithelial-mesenchymal transition (EMT). METHODS AND RESULTS: We used immunohistochemistry to analyse the expression of cytokeratin, E-cadherin, ß-catenin, vimentin, fibronectin, Snail1, Slug and aldehyde dehydrogenase 1 (ALDH1) in 115 cases of NPC in which there were neoplastic spindle cells; in 47 cases a neoplastic squamous cell component was also present. There was no significant difference in the expression of cytokeratin observed in the neoplastic spindle cells (P = 0.644), compared to the squamous component whereas E-cadherin expression was reduced. By contrast, the expression of ß-catenin, vimentin, fibronectin, Snail1, Slug and ALDH1 was up-regulated in the spindle cells (all P = 0.000). Furthermore, E-cadherin expression was associated negatively with ß-catenin (P < 0.001), vimentin (P < 0.001), fibronectin (P < 0.001), Slug (P < 0.001) and ALDH1 (P < 0.001) in neoplastic spindle cells, but did not correlate with Snail1 expression (P = 0.093). CONCLUSIONS: Our findings demonstrate for the first time that EMT might play an important role in the development of neoplastic spindle cells in NPC.

Effects of indirect actions and oxygen on relative biological effectiveness: estimate of DSB inductions and conversions induced by therapeutic proton beams.

Purpose: This study evaluated the DNA double strand breaks (DSBs) induced by indirect actions and its misrepairs to estimate the relative biological effectiveness (RBE) of proton beams.Materials and methods: From experimental data, DSB induction was evaluated in cells irradiated by 62 MeV proton beams in the presence of dimethylsulphoxide (DMSO) and under hypoxic conditions. The DNA damage yields for calculating the RBE were estimated using Monte Carlo Damage Simulation (MCDS) software. The repair outcomes (correct repairs, mutations and DSB conversions) were estimated using Monte Carlo Excision Repair (MCER) simulations.Results: The values for RBE of 62 MeV protons (LET = 1.051 keV/µm) for DSB induction and enzymatic DSB under aerobic condition (21% O2) was 1.02 and 0.94, respectively, as comparing to 60Co γ-rays (LET = 2.4 keV/µm). DMSO mitigated the inference of indirect action and reduced DSB induction to a greater extent when damaged by protons rather than γ-rays, resulting in a decreased RBE of 0.86. DMSO also efficiently prevented enzymatic DSB yields triggered by proton irradiation and reduced the RBE to 0.83. However, hypoxia (2% O2) produced a similar level of DSB induction with respect to the protons and γ-rays, with a comparable RBE of 1.02.Conclusions: The RBE values of proton beams estimated from DSB induction and enzymatic DSB decreased by 16% and 12%, respectively, in the presence of DMSO. Our findings indicate that the overall effects of DSB induction and enzymatic DSB could intensify the tumor killing, while alleviate normal tissue damage when indirect actions are effectively interrupted.

Hes1 triggers epithelial-mesenchymal transition (EMT)-like cellular marker alterations and promotes invasion and metastasis of nasopharyngeal carcinoma by activating the PTEN/AKT pathway.

Overexpression of the transcriptional factor Hes1 (hairy and enhancer of split-1) has been observed in numerous cancers, but the precise roles of Hes1 in epithelial-mesenchymal transition (EMT), cancer invasion and metastasis remain unknown. Our current study firstly revealed that Hes1 upregulation in a cohort of human nasopharyngeal carcinoma (NPC) biopsies is significantly associated with the EMT, invasive and metastatic phenotypes of cancer. In the present study, we found that Hes1 overexpression triggered EMT-like cellular marker alterations of NPC cells, whereas knockdown of Hes1 through shRNA reversed the EMT-like phenotypes, as strongly supported by Hes1-mediated EMT in NPC clinical specimens described above. Gain-of-function and loss-of-function experiments demonstrated that Hes1 promoted the migration and invasion of NPC cells in vitro. In addition, exogenous expression of Hes1 significantly enhanced the metastatic ability of NPC cells in vivo. Chromatin immunoprecipitation (ChIP) assays showed that Hes1 inhibited PTEN expression in NPC cells through binding to PTEN promoter region. Increased Hes1 expression and decreased PTEN expression were also observed in a cohort of NPC biopsies. Additional studies demonstrated that Hes1-induced EMT-like molecular changes and increased motility and invasion of NPC cells were mediated by PTEN. Taken together, our results suggest, for what we believe is the first time, that Hes1 plays an important role in the invasion and metastasis of NPC through inhibiting PTEN expression to trigger EMT-like phenotypes.

Cancer stem cell characteristics, ALDH1 expression in the invasive front of nasopharyngeal carcinoma.

The invasive tumor front underlies the biological aggressiveness and epithelial-mesenchymal transition (EMT) in various human malignances. However, the molecular and biological characteristics of invasive tumor front in NPC have rarely been described. Additionally, the features of cancer stem cells (CSCs) in the invasive front of tumors and its correlation with EMT also remain elusive. Our study was to investigate the expression of CSCs marker aldehyde dehydrogenase 1 (ALDH1) in the invasive front of nasopharyngeal carcinoma (NPC) and its clinical significance. Immunohistochemistry was mainly used to detect ALDH1 expression in the invasive front of NPC. The relationship between ALDH1 expression and EMT-associated markers was also examined. ALDH1 expression in the invasive front correlated strongly with lymphatic invasion (p<0.001), T classification (p=0.001), M classification (p<0.001), clinical stage (p<0.001), and local recurrence (p=0.008). ALDH1 overexpression in the invasive front contributed to worse survival of NPC, particularly in patients with early stage (T1-T2 or N0-N1) (p<0.001 and p=0.002, respectively), though it was not an independent prognostic factor (p=0.196). Furthermore, in the invasive front of NPC, ALDH1 expression correlated significantly with EMT-related biomarkers E-cadherin (p=0.026), Vimentin (p<0.001), Periostin (p<0.001), and Snail (p<0.001), but not with ß-catenin (p=0.143). Our findings demonstrate first that ALDH1 expression in the invasive front links closely with EMT characteristics and tumor aggressiveness, which might provide a useful prognostic marker for NPC patients.

Heparanase overexpression participates in tumor growth of cervical cancer in vitro and in vivo.

Expression of heparanase is associated with invasion, metastasis and angiogenesis of a variety of human cancers. However, the roles of heparanase in cervical cancer are not clear. The aim of this study is to determine whether up-regulation of heparanase expression can promote growth of cervical cancer in vitro and in vivo. Heparanase protein expression was analyzed in cervical cancer patients using immunohistochemistry. In addition, expression of heparanase was also examined in cervical cancer cell lines. A series of in vivo and in vitro assays was performed to elucidate the role of heparanase in tumor growth of cervical cancer. Positive rate of heparanase was 63.3 % (38/60) in cervical cancer patients by immunohistochemistry, and it was significantly correlated with tumor size and clinical stage (P < 0.05). Overexpression of heparanase inhibited apoptosis of cervical cancer cells. Moreover, ectopic overexpression of heparanase in cervical cancer cells promoted proliferation of cervical cancer cells in vitro and tumor growth in vivo. These results suggest that heparanase participates in tumor growth of cervical cancer by influencing the proliferation and apoptosis of cervical cancer cells, and heparanase could be used as an effective therapeutic target for cervical cancer.

[Influence of celecoxib on invasiveness of human high-metastatic nasopharyngeal carcinoma cell line CNE-2Z].

OBJECTIVE: To investigate the effect and mechanism (a selective cyclooxygenase-2 inhibitor) on invasive ability of human nasopharyngeal carcinoma (NPC) line CNE-2Z. METHODS: The proliferation of NPC cells was examined by MTT assay. The invasive and migrating ability of NPC cells was detected with transwell chamber. E-cadherin protein expression was detected by immunocytochemistry and the expressions of Cox-2 and E-cadherin mRNA were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: MTT showed that celecoxib inhibited CNE-2Z proliferation in dose-dependent manner, the survival rate of cells treated with 25, 50, 100 µmol/L celecoxib (x(-) ± s) for 24 h was (94.75 ± 1.34)%, (91.77 ± 2.70)%, (64.54 ± 1.20)%, respectively, and the survival rate of cells treated for 48 h was (88.41 ± 1.28)%, (78.84 ± 1.56)%, (52.46 ± 2.25)%, respectively, the concentration of 50% inhibition concentration of a substance (IC50) was 100 µmol/L, the difference was statistically significant between different concentration groups in the same time-point (respectively, F were 462.204 and 1328.306, P < 0.01). Treated with different concentrations of celecoxib (0, 25, 50 µmol/L) for 24, the cell numbers (x(-) ± s) through PVPF by tumor invasion assay were (263.7 ± 13.5), (185.3 ± 8.7) and (144.0 ± 8.2), the difference was statistically significant between the experimental and control group (F = 102.089, P < 0.01). Immunocytochemistry showed that celecoxib significantly induced the increase of E-cadherin protein expression, also with a dose-dependence in 0 µmol/L, 25 µmol/L, 50 µmol/L group was (21.7 ± 2.6), (28.7 ± 2.4), (40.3 ± 1.3), and 50 µmol/L group increased significantly (F = 78.637, P < 0.01). RT-PCR showed that celecoxib reduced the expression of Cox-2 mRNA expression in 25, 50 µmol/L group decreased significantly compared with the control group (respectively, t were 23.950 and 36.651, P < 0.01), but it enhanced the expression of E-cadherin mRNA expression in 25, 50 µmol/L group was significantly higher (respectively, t were 35.829 and 81.497, P < 0.01). CONCLUSION: Celecoxib can inhibits the invasive ability of NPC cell line CNE-2Z, which possibly relates with the upregulated expression of E-cadherin.

[Effects of Epstein-Barr virus latent membrane protein 1 on metastasis of human nasopharyngeal carcinoma cell lines].

BACKGROUND & OBJECTIVE: Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) plays an important role in the metastasis of nasopharyngeal carcinoma (NPC). This study was to investigate the effects of EBV LMP1 on the metastasis of NPC cell lines, and explore potential mechanism. METHODS: The expression of LMP1, E-cadherin and intercellular adhesion molecule-1 (ICAM-1) in human NPC cell lines CNE1 (well differentiated) and CNE1-GL (CNE1 cells transfected with LMP1) were detected by SP immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The cell-cell adhesion assay, the cell-matrix adhesion assay, the wound-induced migration assay and the migration assay were used to investigate the effects of LMP1 on adhesive and metastatic abilities of NPC cells. RESULTS: The positive rates of LMP1 and ICAM-1 were significantly lower in CNE1 cells than in CNE1-GL cells [0% vs. (96.60+/-3.03)%, P<0.01; (5.27+/-1.45)% vs. (93.33+/-4.23)%, P<0.01]; the positive rate of E-cadherin was significantly higher in CNE1 cells than in CNE1-GL cells [(37.47+/-1.50)% vs. (19.53+/-1.92)%, P<0.01]. The expression of E-cadherin was significantly inhibited (P<0.01), while the expression of ICAM-1 was significantly increased (P<0.01) in CNE1-GL cells as compared with those in CNE1 cells. The cell-cell adhesive ability of CNE1-GL cells was lower than that of CNE1 cells (P<0.05). The cell-matrix adhesive ability of CNE1-GL cells was significantly higher than that of CNE1 cells (0.60+/-0.03 vs. 0.46+/-0.01, P<0.01). The number of migrated CNE1-GL cells was higher than that of migrated CNE1 cells (119.3+/-6.0 vs. 46.3+/-7.0, P<0.05). CONCLUSION: By inhibiting E-cadherin expression and enhancing ICAM-1 expression, LMP1 may reduce the cell-cell adhesive ability and improve the cell-matrix adhesive ability and migratory ability of NPC cells, which may play roles in the invasion and metastasis of NPC.