Histone deacetylases inhibitor MS-275 suppresses human esophageal squamous cell carcinoma cell growth and progression via the PI3K/Akt/mTOR pathway

Esophageal squamous cell carcinoma (ESCC) is a malignant tumor with low survival rate, so new therapies are urgently needed. Histone deacetylases (HDACs) play a critical role in tumorigenesis, and HDACs inhibition is a potential therapeutic target in ESSC. In our study, we evaluated the effect and molecular mechanism of MS-275 (an inhibitor of HDACs) on ESCC cells. We found that HDAC1 and HDAC2 were overexpressed in ESCC tissues and related with clinical pathological features of patients with ESCC. MS-275 markedly reduced HDAC1 and HDAC2 expression, whereas increased the level of AcH3 and AcH2B. MS-275 suppressed proliferation and clonogenicity of ESCC cells in a concentration-dependent manner. In addition, MS-275 induced apoptosis, arrested cell cycle, and inhibited migration, epithelial–mesenchymal transition, and sphere-forming ability of ESCC cells in vitro. Moreover, p-Akt1 and p-mTOR were downregulated by MS-275. Finally, MS-275 significantly inhibited tumor growth in vivo. Taken together, HDAC1 and HDAC2 are associated with the progression of ESCC, and MS-275 hinders the progression and stemness of ESCC cells by suppressing the PI3K/Akt/mTOR pathway. Our findings show that MS-275 inhibits ESCC cells growth in vitro and in vivo, which is a potential drug for the ESCC therapy.     

Expression and activity of mTOR and its substrates in different cell cycle phases and in oral squamous cell carcinomas of different malignant grade

In order to study the relationship between mTOR (mammalian target of rapamycin) and tumorigenesis, we investigated the expression and activity of mTOR, and its substrates, alpha1, alpha2, beta1 and beta2 isoforms of p70S6 kinase (p70S6K) and eukaryotic initiation factor 4E binding protein-1 (4EBP-1) in oral squamous carcinoma and Hela cells using RT-PCR, immunohistochemistry, statistical analysis and Western blotting. The result of Western blots showed that in poorly differentiated oral squamous carcinoma, the expression level of mTOR and p70S6k increased in M phase, while that of 4EBP-1 decreased. The results of RT-PCR and immunohistochemistry assay are the same as that of Western blot. In Hela cells, the RT-PCR results showed that the level of mTOR mRNA did not change during the cell cycle. In M phase, the expression of alpha1, alpha2, beta1 and beta2 isoforms of p70S6K increased noticeably, while the expression of 4EBP-1 decreased. The immunoblot results in Hela cells were consistent with the RT-PCR results. Furthermore, the activity assays in Hela cells suggested that,in phase G2 and M, the activity of mTOR was maintained at a higher level than in any other phase, while 4EBP-1 decreased in phase M. These results may help in further investigations of the important role of mTOR in cell cycle and tumorigenesis.     

Cytotoxic T cell responses to Streptococcus are associated with improved prognosis of oral squamous cell carcinoma

Several species of Streptococcus, such as S. salivarius, S. mitis, and S. anginosus, are found to extensively colonize the oral cavity and the upper respiratory tract, and have been shown to increase in patients with oral squamous cell carcinoma (OSCC). Accumulating evidence have revealed that commensal bacteria are involved in antitumor immunity via T cell-mediated mechanisms, but the role of Streptococcus enrichment in OSCC is yet unclear. In this study, we stimulated peripheral blood mononuclear cells from non-cancer controls (NCs) and OSCC patients with S. salivarius, S. mitis, and S. anginosus. We observed that compared to NC subjects, OSCC patients at earlier stages had higher frequencies of granzyme B-expressing CD8 T cells for all Streptococcus species tested, while OSCC patients at more advanced stages had higher frequencies of granzyme B-expressing CD8 T cells for S. anginosus but not other Streptococcus species. In OSCC patients, the Streptococcus-reactive CD8 T cells presented significantly lower levels of PD-1 and TIM-3 expression than Streptococcus-nonreactive CD8 T cells. The clinical outcomes of OSCC patients in our cohort were tracked for 24 months after the resection of the primary tumor. In patients that did not present tumor recurrence, the frequencies of S. salivarius-reactive and S. mitis-reactive CD8 T cells were significantly higher than that in patients that developed recurrent tumor. Furthermore, in patients with tumor recurrence, the duration between primary tumor resection and tumor recurrence was positively associated with the frequencies of S. salivarius-reactive and S. anginosus-reactive CD8 T cells. Together, we demonstrated that Streptococcus-reactive CD8 T cell responses might contribute to antitumor immunity in OSCC patients.     

Circular RNA circ-EGLN3 promotes renal cell carcinoma proliferation and aggressiveness via miR-1299-mediated IRF7 activation

Renal cell carcinoma (RCC) is a highly lethal cancer with increasing incidence worldwide. The purpose of the present study was to investigate the functions and molecular mechanisms of circular RNA (circRNA), circ-EGLN3, in RCC progression. The expression levels of circ-EGLN3 were assessed by a quantitative real-time polymerase chain reaction. Kaplan-Meier analysis was applied to uncover the prognostic role of circ-EGLN3 in patients with RCC. Cell viability was analyzed using cell counting kit-8 and cell apoptosis was assessed using flow cytometric experiment. Cell migratory and invasive abilities were determined by wound scratch and transwell experiments. Subcellular distribution detection was utilized to investigate the location of circ-EGLN3. Dual-luciferase reporter test was utilized for identifying the molecular mechanism of circ-EGLN3. The results indicated that circ-EGLN3 was elevated in RCC tissues and cell lines and predicted unfavorable prognosis for the patients with RCC. Silenced circ-EGLN3 hindered cell proliferation, migration, and invasion but facilitated apoptosis of RCC cells. Ectopic expressed circ-EGLN3 induced the opposite effects mentioned above. Mechanistically, circ-EGLN3 was mainly located at the cytoplasm. Circ-EGLN3 acted as a competing endogenous RNA (ceRNA) to enhance the IRF7 level via sponging miR-1299. Moreover, circ-EGLN3 mediated elevation of IRF7 is responsible for RCC cell proliferation and aggressiveness. Collectively, our study suggested that circ-EGLN3 knockdown suppressed RCC progression through acting as a ceRNA to regulate the IRF7 expression by targeting miR-1299. Circ-EGLN3 might be a potential therapeutic target for RCC management.     

Reversine suppresses oral squamous cell carcinoma via cell cycle arrest and concomitantly apoptosis and autophagy

Background

The effective therapies for oral cancer patients of stage III and IV are generally surgical excision and radiation combined with adjuvant chemotherapy using 5-Fu and Cisplatin. However, the five-year survival rate is still less than 30% in Taiwan. Therefore, evaluation of effective drugs for oral cancer treatment is an important issue. Many studies indicated that aurora kinases (A, B and C) were potential targets for cancer therapies. Reversine was proved to be a novel aurora kinases inhibitor with lower toxicity recently. In this study, the potentiality for reversine as an anticancer agent in oral squamous cell carcinoma (OSCC) was evaluated.

Methods

Effects of reversine on cell growth, cell cycle progress, apoptosis, and autophagy were evaluated mainly by cell counting, flow cytometry, immunoblot, and immunofluorescence.

Results

The results demonstrated that reversine significantly suppressed the proliferation of two OSCC cell lines (OC2 and OCSL) and markedly rendered cell cycle arrest at G2/M stage. Reversine also induced cell death via both caspase-dependent and -independent apoptosis. In addition, reversine could inhibit Akt/mTORC1 signaling pathway, accounting for its ability to induce autophagy.

Conclusions

Taken together, reversine suppresses growth of OSCC via multiple mechanisms, which may be a unique advantage for developing novel therapeutic regimens for treatment of oral cancer in the future.     

Nucleolar spindle-associated protein 1 promotes tumorigenesis and predicts poor prognosis in human esophageal squamous cell carcinoma

The microtubule binding protein, nucleolar spindle-associated protein 1 (NUSAP1), has a crucial function in mitosis and its expression is closely associated with carcinogenesis. Herein, we aimed to determine the function of NUSAP1 in the development of human esophageal squamous cell carcinoma (ESCC), and the association of NUSAP1 expression with ESCC. Immunohistochemical staining of ESCC tissue sections indicated that NUSAP1 was expressed to a higher degree in tumor tissues than in adjacent nontumor tissues. NUSAP1 levels were relevant closely to histological differentiation (P = 0.049). Overall survival was longer in patients with lower NUSAP1 levels ( P < 0.001). NUSAP1 expression ( P = 0.002), histological differentiation ( P < 0.001), tumor depth ( P = 0.045), lymph node metastases ( P < 0.001), and tumor-node-metastasis staging ( P = 0.008) were greatly associated with overall survival using univariate analysis. Multivariate analysis suggested that histological differentiation ( P = 0.014) and NUSAP1 expression ( P = 0.026) could be independent prognostic markers for ESCC. Additionally, the biological behavior of ESCC cells was investigated in vitro and in vivo. Suppression of NUSAP1 inhibited cellular proliferation and invasion, and induced cell cycle arrest and apoptosis in vitro. More importantly, knockdown of NUSAP1 led to inhibition of tumor formation in nude mice. These findings indicated that NUSAP1 is a potential prognostic biomarker in ESCC, and is an ESCC oncogene. Thus, NUSAP1 could represent a therapeutic target for ESCC.     

Circular RNA circMDM2 accelerates the glycolysis of oral squamous cell carcinoma by targeting miR‐532‐3p/HK2

Circular RNAs (circRNAs) function as an essential regulator in the progression of oral squamous cell carcinoma (OSCC). However, the potential roles and mechanism of circRNAs in OSCC are still elusive. Here, this research investigates the roles and molecular mechanism of novel circRNA (circMDM2) in OSCC progression. Clinically, circMDM2 was overexpressed in OSCC tissue and cells, and the overexpression served as a poor prognostic factor for OSCC patients. Functionally, cellular experiments confirmed that circMDM2 accelerated OSCC cell proliferation and glycolysis in vitro and circMDM2 knockdown repressed the tumour growth in vivo. Mechanistically, circMDM2 sponged miR‐532‐3p to promote the hexokinase 2 (HK2), forming the circMDM2/miR‐532‐3p/HK2 axis. In conclusion, these findings demonstrated that circMDM2/miR‐532‐3p/HK2 axis promotes the proliferation and glycolysis of OSCC, rendering a potential diagnostic biomarker and prospective therapeutic target for OSCC.     

Circ_0000052/miR-382-3p axis induces PD-L1 expression and regulates cell proliferation and immune evasion in head and neck squamous cell carcinoma

A better understanding of the mechanisms underlying PD-L1 aberrant expression in head and neck squamous cell carcinoma (HNSCC) will help reveal predictive biomarkers and overcome resistance to treatment. In this study, the prognostic significance of PD-L1 in forty-five HNSCC archival samples was determined by qRT-PCR. The biological function associated with malignant behaviour was assessed by PD-L1 depletion, miR-382-3p re-expression and regulation of circ_0000052. The interactions of PD-L1-miRNA and miRNA-circRNA were determined by qRT-PCR, Western blot analysis, dual-luciferase reporter assays and RNA immunoprecipitation assays. PD-L1 was highly expressed in patient samples and cancer cell lines. Higher levels of PD-L1 were associated with patient recurrences and play a pivotal role in regulating cell proliferation, migration, invasion, clonogenicity and apoptosis. In addition to demonstrating that the IFN-γ/JAK2/STAT1 signalling pathway can induce PD-L1 overexpression in HNSCC, a novel mechanism by which upregulated circ_0000052 mediates PD-L1 overexpression was also demonstrated. To do this, circ_0000052 competitively binds to miR-382-3p and alleviates its repression of PD-L1. This leads to overexpression of PD-L1, causing the aggressiveness of the cells. Our data demonstrate that circ_0000052 is oncogenic, and the circ_0000052/miR-382-3p/PD-L1 axis is critical in HNSCC progression. The manipulation of circRNAs/miRNAs in combination with anti-PD-L1 therapy may improve personalized disease management.     

Increased salivary microvesicles are associated with the prognosis of patients with oral squamous cell carcinoma

Microvesicles (MVs), which are cell‐derived membrane vesicles present in body fluids, are closely associated with the development of malignant tumours. Saliva, one of the most versatile body fluids, is an important source of MVs. However, the association between salivary MVs (SMVs) and oral squamous cell carcinoma (OSCC), which is directly immersed in the salivary milieu, remains unclear. SMVs from 65 patients with OSCC, 21 patients with oral ulcer (OU), and 42 healthy donors were purified, quantified and analysed for their correlations with the clinicopathologic features and prognosis of OSCC patients. The results showed that the level of SMVs was significantly elevated in patients with OSCC compared to healthy donors and OU patients. Meanwhile, the level of SMVs showed close correlations with the lymph node status, and the clinical stage of OSCC patients. Additionally, the ratio of apoptotic to non‐apoptotic SMVs was significantly decreased in OSCC patients with higher pathological grade. Consistently, poorer overall survival was observed in patients with lower ratio of apoptotic to non‐apoptotic SMVs. In conclusion, the elevated level of SMVs is associated with clinicopathologic features and decreased survival in patients with OSCC, suggesting that SMVs are a potential biomarker and/or regulator of the malignant progression of OSCC.     

TUSC3 inhibits cell proliferation and invasion in cervical squamous cell carcinoma via suppression of the AKT signalling pathway

The decreased expression of tumour suppressor candidate 3 (TUSC3) is associated with proliferation in several types of cancer, leading to an unfavourable prognosis. The present study aimed to assess the cellular and molecular function of TUSC3 in patients with cervical squamous cell carcinoma (CSCC). Levels of mRNA expressions of TUSC3 were analysed in CSCC tissues and six cell lines using qRT‐PCR. Immunohistochemistry(IHC) was used to evaluate the protein expression level of TUSC3 in four paired specimens, 220 paraffin‐embedded CSCC specimens and 60 cases of normal cervical tissues(NCTs), respectively. Short hairpin RNA interference was employed for TUSC3 knockdown. Cell proliferation, migration and invasion were evaluated using growth curve, MTT assay, wound healing, transwell assay and xenograft tumour model, respectively. The results demonstrated that TUSC3 mRNA and protein expression levels were downregulated in CSCC samples. Multivariate and univariate analyses indicated that TUSC3 was an independent prognostic factor for patients with CSCC. Decreased TUSC3 expression levels were significantly associated with proliferation and an aggressive phenotype of cervical cancer cells both in vitro and in vivo. Moreover, the knockdown of TUSC3 promoted migration and invasion of cancer cells, while the increased expression of TUSC3 exhibited the opposite effects. The downregulation of TUSC3 facilitated proliferation and invasion of CSCC cells through the activation of the AKT signalling pathway. Our data demonstrated that the downregulation of TUSC3 promoted CSCC cell metastasis via the AKT signalling pathway. Therefore, TUSC3 may serve as a novel prognostic marker and potential target for CSCC.     

Altered serum and salivary C-reactive protein levels in patients with oral premalignant lesions and oral squamous cell carcinoma

Chronic inflammation is associated with cancer development. C-reactive protein (CRP), an acute phase protein synthesized primarily in the liver, is a marker for inflammation and for the progression of many cancers. We compared serum and salivary CRP levels in 20 normal individuals, 20 patients with oral premalignant lesions and 20 patients with oral squamous cell carcinoma (OSCC) to assess its efficacy as a prognostic indicator for OSCC. Saliva and blood samples were obtained and evaluated for CRP levels. Mean CRP levels were higher in patients with oral premalignant lesions compared to controls. CRP levels in OSCC patients were elevated and were associated with advanced tumor stages.     

NUMB在口腔白斑和鳞癌中的表达变化及意义

目的:研究NUMB在口腔白斑和鳞癌中的表达及意义。方法:采用免疫组织化学法检测88例口腔正常黏膜、口腔白斑及口腔鳞癌石蜡包埋组织中NUMB蛋白的表达。结果:NUMB在口腔正常黏膜(95.7%)、口腔白斑(75%)及口腔鳞癌(4.7%)中均有阳性表达但是表达频率依次降低。NUMB在各个组中的阳性表达率具有统计学差异(P0.05)。结论NUMB阳性表达率随口腔组织恶性程度增高而减少的趋势提示该基因可能在口腔正常黏膜到口腔白斑再到口腔鳞癌的转化中起作用,NUMB有可能成为早期发现癌变的分子生物学标志之一。