Wantao Chen

Loss of microRNA-143/145 disturbs cellular growth and apoptosis of human epithelial cancers by impairing the MDM2-p53 feedback loop

Dysregulated microRNAs (miRNAs) have an important role in many malignant tumors. However, elucidating the roles of miRNAs in cancer biology, especially in epithelial cancers, remains an ongoing process. In this study, we show that both miR-143 and miR-145, which belong to the same miRNA cluster, can negatively modulate expression of their target gene, MDM2. The miR-143 and miR-145 is posttranscriptionally activated by upregulated p53, thereby generating a short miRNAs-MDM2-p53 feedback loop. Re-expression of these miRNAs suppresses cellular growth and triggers the apoptosis of epithelial cancer, in vitro and in vivo, by enhancing p53 activity via MDM2 turnover. Moreover, the miRNA-dependent MDM2 turnover contributes to the equilibrium of repeated p53 pulses in response to DNA damage stress. These findings suggest that MDM2 dysregulation caused by downregulation of miR-143 and miR-145 contributes to epithelial cancer development and has a key role in regulating cellular proliferation and apoptosis. Re-expression of miR-143 and miR-145 may be a reasonable strategy for treatment of epithelial cancers.

Side population in oral squamous cell carcinoma possesses tumor stem cell phenotypes

To characterize the biological features of side population (SP) in oral squamous cell carcinoma (OCC), SP and non-SP were sorted and compared. The SP cells were more clonogenic and in nude mice, only 10,000 SP cells were needed for tumor development compared to 1,000,000 non-SP cells. The SP cells expressed higher levels of ABCG2, ABCB1, CD44, Oct-4, Bmi-1, NSPc1 and CK19. The SP cells generated SP and non-SP populations, whereas the non-SP cells generated only non-SP. These findings provide the first evidence that SP in OCC possesses tumor stem cell phenotypes and may play an important role in OCC tumorigenesis.

Selection and validation of differentially expressed genes in head and neck cancer.

We applied a robust combinatorial (multi-test) approach to microarray data to identify genes consistently up- or down-regulated in head and neck squamous cell carcinoma (HNSCC). RNA was extracted from 22 paired samples of HNSCC and normal tissue from the same donors and hybridized to the Affymetrix U95A chip. Forty-two differentially expressed probe sets (representing 38 genes and one expressed sequence tag) satisfied all statistical tests of significance and were selected for further validation. Selected probe sets were validated by hierarchical clustering, multiple probe set concordance, and target-subunit agreement. In addition, real-time PCR analysis of 8 representative (randomly selected from 38) genes performed on both microarray-tested and independently obtained samples correlated well with the microarray data. The genes identified and validated by this method were in comparatively good agreement with other rigorous HNSCC microarray studies. From this study, we conclude that combinatorial analysis of microarray data is a promising technique for identifying differentially expressed genes with few false positives.

MicroRNAs contribute to the chemoresistance of cisplatin in tongue squamous cell carcinoma lines

MicroRNAs (miRNAs) are small non-coding RNAs that function as negative regulators of gene expression. They are strongly implicated in human cancers, including oral squamous cell carcinoma (OSCC). Evidence for the involvement of miRNAs as important regulators of chemosensitivity and chemoresistance in OSCC is not well understood. In this study, miRNA microarray was firstly used to compare the differential miRNAs levels between the cisplatin-sensitive tongue squamous cell carcinoma line (Tca8113) and its cisplatin-resistant subline (Tca/cisplatin). Three miRNAs of miR-21, -214, and -23a were validated by miRNAs real-time PCR, and intervened by anti-miRNA oligonucleotides (miR-214 and -23a) and pre-miRNA plasmid transfection (miR-21). Further relationship between miR-23a and DNA topoisomerase II beta (TOP2B) on the chemoresistance against cisplatin was studied. There were 19 out of 480 differential miRNAs between the Tca8113 and Tca/cisplatin cells. miR-214 and -23a were found increased as with chemoresistance against cisplatin in the Tca/cisplatin cells while miR-21 was found decreased as with chemosensitivity for cisplatin in the Tca/cisplatin cells. Intervention of these three miRNAs could decrease the chemoresistance against cisplatin in Tca/cisplatin cells. Transfection of anti-miR-23a into the Tca/cisplatin cells could increase the TOP2B protein expression. Our results suggest the existence of differential miRNAs with chemosensitivity and chemoresistance between the cisplatin-sensitive and resistant tongue squamous cell carcinoma lines. miR-21 serves as a chemosensitive miRNA, while miR-214 and -23a serve as chemoresistant miRNAs in the tongue squamous cell carcinoma lines. miR-23a is an up-stream regulator of TOP2B to realize the chemoresistance of cisplatin.

Growth inhibition and induction of apoptosis in human oral squamous cell carcinoma Tca‐8113 cell lines by shikonin was partly through the inactivation of NF‐κB pathway

Shikonin, a naphthoquinone pigment isolated from the Chinese herbal therapeutic, Zicao, has been shown to exhibit antioxidant and anticancer effects. In this study, its ability to induce apoptosis in cultured Tca-8113 oral cancer cells was studied. Treatment of the Tca-8113 cells with a variety of concentrations of Shikonin (10-40 microm) resulted in dose- and time-dependent sequences of events marked by apoptosis, as shown by the loss of cell viability, chromatin condensation, internucleosomal DNA fragmentation and sub-G1 phase accumulation. Furthermore, apoptosis in the Tca-8113 cells was accompanied by the activation of protease caspase-8, -9, -3 and low expression of Bcl-2 protein. Interestingly, inactivation of the NF-kappaB pathway was found in shikonin-induced apoptosis in Tca-8113 cells. These results raise the possibility that the anti-tumor effects of Shikonin in Tca-8113 cells are at least partly through the inactivation of the NF-kappaB pathway and subsequent activation of protease caspase family. Pharmacological inhibition of the NF-kappaB activity by Shikonin might be a powerful treatment option for OSCC in which activation of NF-kappaB plays a critical role in tumor growth and progression.

The NF-kappa B inhibitor, celastrol, could enhance the anti-cancer effect of gambogic acid on oral squamous cell carcinoma.

BackgroundGambogic acid (GA) is a major active ingredient of gamboge, a widely used traditional Chinese medicine that has been reported to be a potent cytotoxic agent against some malignant tumors. Many studies have shown that the NF-kappa B signaling pathway plays an important role in anti-apoptosis and the drug resistance of tumor cells during chemotherapy. In this study, the effects and mechanisms of GA and the NF-kappa B inhibitor celastrol on oral cancer cells were investigated.MethodsThree human oral squamous cell carcinoma cell lines, Tca8113, TSCC and NT, were treated with GA alone, celastrol alone or GA plus celastrol. Cytotoxicity was assessed by MTT assay. The rate of apoptosis was examined with annexin V/PI staining as well as transmission electronic microscopy in Tca8113 cells. The level of constitutive NF-kappa B activity in oral squamous cell carcinoma cell lines was determined by immunofluorescence assays and nuclear extracts and electrophoretic mobility shift assays (EMSAs) in vitro. To further investigate the role of NF-kappa B activity in GA and celastrol treatment in oral squamous cell carcinoma, we used the dominant negative mutant SR-IκBα to inhibit NF-kappa B activity and to observe its influence on the effect of GA.ResultsThe results showed that GA could inhibit the proliferation and induce the apoptosis of the oral squamous cell carcinoma cell lines and that the NF-kappa B pathway was simultaneously activated by GA treatment. The minimal cytotoxic dose of celastrol was able to effectively suppress the GA-induced NF-kappa B pathway activation. Following the combined treatment with GA and the minimal cytotoxic dose of celastrol or the dominant negative mutant SR-IκBα, proliferation was significantly inhibited, and the apoptotic rate of Tca8113 cells was significantly increased.ConclusionThe combination of GA and celastrol has a synergistic antitumor effect. The effect can be primarily attributed to apoptosis induced by a decrease in NF-kappa B pathway activation. The NF-kappa B signaling pathway plays an important role in this process. Therefore, combining GA and celastrol may be a promising modality for treating oral squamous cell carcinoma.

Correlation of NF-κB signal pathway with tumor metastasis of human head and neck squamous cell carcinoma

BackgroundNuclear factor-kappa B (NF-κB) signaling constitutes a key event in the multistep process of carcinogenesis, progression and treatment in many cancer types. However, the significance of NF-κB pathway for complex and tissue-specific aspects of head and neck cancer progression, such as invasion and metastasis, is less understood.MethodsThe expression of NF-κB p65 in squamous cell carcinoma of the head and neck (SCCHN) clinical specimens by immunohistochemistry. The role of NF-κB activity in head and neck squamous cell carcinoma was determined by western blot, reporter assay and EMSA analysis in vitro and metastasis assays in vivo in different metastatic potential tumor cells. Furthermore, the apoptosis rate and expression of metastasis-related protein such as MMP9 and VEGF were examined by Annexin V/PI staining and Western blot, respectively.ResultsA higher level of active nuclear-localized NF-κB was observed in the metastatic SCCHN specimens group (p < 0.01). The NF-κB activities of SCCHN cell lines with different metastatic potentials were then determined and in excellent agreement with results found in SCCHN specimens, highly metastatic SCCHN cell lines expressed high level of NF-κB activity. The treatment of highly metastatic SCCHN cells with NF-κB inhibitors reduced the in vitro cell invasion capacity of the cells without affecting the apoptotic rate. Additionally, the NF-κB inhibitors significantly inhibited the experimental lung metastasis of Tb cells and lymph node metastasis of TL cells in nude mice. Furthermore, the expression of metastasis-related proteins, such as matrix metalloproteinase 9 and vascular endothelial growth factor, was inhibited by pyrrolidine dithiocarbonate.ConclusionsThis study suggests that NF-κB activity significantly contributes to tumor hematologic and lymphatic metastases and may aid in the development of early detection methods or therapies targeting non-conventional molecular targets.

Dysregulated miR-363 affects head and neck cancer invasion and metastasis by targeting podoplanin.

Head and neck squamous cell carcinoma (HNSCC) is characterised by an elevated capacity for tumour invasion and lymph node metastasis and the cause remains to be determined. Recent studies suggest that microRNAs can regulate the evolution of malignant behaviours by regulating multiple target genes. In this study, we have first confirmed that miR-363 is down-regulated in HNSCC tissues with lymph node metastasis and cell lines with highly invasive capacity. We used bioinformatics, cellular and molecular methods to predict and prove that miR-363 directly targeted to podoplanin (PDPN) and caused up-regulation of PDPN in HNSCC. MSP assay showed that DNA promoter methylation was involved in silencing the miR-363 in HNSCC. Furthermore, we provided evidence to demonstrate that PDPN dysregulation caused by down-regulation of miR-363 contributes to HNSCC invasion and metastasis. These data reveal a key role of miR-363-PDPN in HNSCC metastasis and support biological and clinical links between miR-363-PDPN and HNSCC.

MiR-145, a new regulator of the DNA Fragmentation Factor-45 (DFF45)-mediated apoptotic network

BackgroundMicroRNA-145 (miR-145) is considered to play key roles in many cellular processes, such as proliferation, differentiation and apoptosis, by inhibiting target gene expression. DNA Fragmentation Factor-45 (DFF45) has been found to be the substrate of Caspase-3, and the cleavage of DFF45 by caspase-3 during apoptosis releases DFF40 that degrades chromosomal DNA into nucleosomal fragments. There are currently no in-depth studies on the relationship between miR-145 and the DFF45 gene.ResultsIn this study, we identified DFF45 as a novel target of miR-145. We demonstrated that miR-145 targets a putative binding site in the coding sequence (CDS) of DFF45, and its abundance is inversely associated with DFF45 expression in colon cancer cells. Using a luciferase reporter system, we found that miR-145 suppresses the expression of the luciferase reporter gene fused to the putative binding site of DFF45. The level of DFF45 protein, but not DFF45 mRNA, was decreased by miR-145, suggesting a mechanism of translational regulation. Furthermore, we demonstrate that this specific silencing of DFF45 by miR-145 accounts, at least in part, for the staurosporine-induced tumor cell apoptosis in vitro.ConclusionsOur study reveals a previously unrecognized function of miR-145 in DFF45 processing, which may underlie crucial aspects of cancer biology.

Common and Complex Notch1 Mutations in Chinese Oral Squamous Cell Carcinoma

Purpose: To determine Notch1 mutation status in oral squamous cell carcinoma (OSCC) from Chinese population and its potential clinical implications. Experimental Design: Surgically resected OSCC tissues from 51 Chinese patients and 13 head and neck squamous cell carcinoma (HNSCC) cell lines were sequenced for mutations in the entire coding regions of Notch1 and TP53 using a next-generation sequencing platform. Sequences of the genes were also determined in corresponding normal tissues from 46 of the 51 patients. Mutations and their association with clinical parameters were analyzed. Results: Six mutations in Notch1 and 11 mutations in TP53 coding regions were detected in 4 (31%) and 10 (77%) of the 13 HNSCC cell lines, respectively. Forty-two somatic Notch1 mutations, including 7 nonsense mutations and 11 mutations within the domain commonly harboring potential activating mutations in acute lymphoblastic leukemia, were detected in 22 (43%) of the 51 Chinese OSCC tumors. In comparison, 25 somatic TP53 mutations were observed in 21 (41%) of the 51 tumors. Patients whose tumors carried Notch1 mutation had significantly shorter overall and disease-free survivals (P = 0.004 and P = 0.001, respectively, by log-rank test) compared with those whose tumors carried no Notch1 mutation. Multivariate analysis showed that both Notch1 mutation and lymph node metastasis are independent prognostic factors in the patient population (P = 0.001). All 15 patients with both Notch1 mutation and nodal metastasis recurred or metastasized within 2 years after surgery. Conclusions: Notch1 mutation is common in Chinese OSCC and associates with clinical outcomes. The complexity of the mutation spectrum warrants further investigation of Notch1 in Chinese patients with OSCC. Clin Cancer Res; 20(3); 701–10. ©2013 AACR.