Quentin Liu

The pivotal role of c-Jun NH2-terminal kinase-mediated Beclin 1 expression during anticancer agents-induced autophagy in cancer cells

The c-Jun NH2-terminal kinase (JNK) pathway represents one subgroup of MAP kinases that are activated primarily by cytokines and exposure to environmental stress. Autophagy is a protein-degradation system characterized by the formation of double-membrane vacuoles termed autophagosomes. Autophagy-related gene beclin 1 plays a key role in autophagosome formation. However, the relationships between activation of JNK pathway, autophagy induction and Beclin 1 expression remain elusive. In this study, we used human cancer cell lines CNE2 and Hep3B to investigate the role of JNK-mediated Beclin 1 expression in ceramide-induced autophagic cell death. Ceramide-treated cells exhibited the characteristics of autophagy (that is, acidic vesicular organelle formation and the LC3-II generation). JNK was activated in these two cell lines exposed to ceramide and the phosphorylation of c-Jun also increased. In the meantime, we found that ceramide upregulated Beclin 1 expression in cancer cells. The upregulation of Beclin 1 expression could be blocked by SP600125 (a specific inhibitor of JNK) or a small interfering RNA (siRNA) directed against JNK1/2 or c-Jun. Chromatin immunoprecipitation and luciferase reporter analysis revealed that c-Jun was involved in the regulation of beclin 1 transcription in response to ceramide treatment. In addition, inhibition of JNK activity by SP600125 could inhibit autophagy induction by ceramide. Furthermore, Beclin 1 knockdown by siRNA also inhibited ceramide-mediated autophagic cell death. JNK-mediated Beclin 1 expression was also observed in topotecan-induced autophagy. These data suggest that activation of JNK pathway can mediate Beclin 1 expression, which plays a key role in autophagic cell death in cancer cells.

MiR-200b and miR-15b regulate chemotherapy-induced epithelial-mesenchymal transition in human tongue cancer cells by targeting BMI1

Chemotherapy has been reported to induce epithelial-mesenchymal transition (EMT) in tumor cells, which is a critical step in the process of metastasis leading to cancer spreading and treatment failure. However, the underlying mechanisms of chemotherapy-induced EMT remain unclear, and the involvement of microRNAs (miRNA) in this process is poorly understood. To address these questions, we established stable chemotherapy-resistant tongue squamous cell carcinoma (TSCC) cell lines CAL27-res and SCC25-res by exposing the parental CAL27 and SCC25 lines to escalating concentrations of cisplatin for 6 months. CAL27-res and SCC25-res cells displayed mesenchymal features with enhanced invasiveness and motility. MiRNA microarray illustrated that miR-200b and miR-15b were the most significantly downregulated microRNAs in CAL27-res cells. Ectopic expression of miR-200b and miR-15b with miRNA mimics effectively reversed the phenotype of EMT in CAL27-res and SCC25-res cells, and sensitized them to chemotherapy, but inhibition of miR-200b and miR-15b in the sensitive lines with anti-sense oligonucleotides induced EMT and conferred chemoresistance. Retrieving the expression of B lymphoma Mo-MLV insertion region 1 homolog (BMI1), a target for miR-200b and miR-15b, in the presence of the miRNA mimics by transfecting CAL27-res cells with pcDNA3.1–BMI1-carrying mutated seed sequences of miR-200b or miR-15b at its 3′-UTR recapitulated chemotherapy-induced EMT. In vivo, enforced miR-200b or miR-15b expression suppressed metastasis of TSCC xenografts established by CAL27-res cells. Clinically, reduced miR-200b or miR-15b expression was associated with chemotherapeutic resistance in TSCCs and poor patient survival. Our data suggest that reduced expression of miR-200b and miR-15b underscores the mechanisms of chemotherapy-induced EMT in TSCC, and may serve as therapeutic targets to reverse chemotherapy resistance in tongue cancers.

Aurora-A, a Negative Prognostic Marker, Increases Migration and Decreases Radiosensitivity in Cancer Cells

Centrosomal Aurora-A (Aur-A) kinase ensures proper spindle assembly and accurate chromosome segregation in mitosis. Overexpression of Aur-A leads to centrosome amplification, aberrant spindle, and consequent genetic instability. In the present study, Aur-A was found to be overexpressed in laryngeal squamous cell carcinoma (LSCC). Moreover, Aur-A expression was adversely correlated with median survival, and further identified as a potential independent factor for disease prognosis. Suppression of Aurora kinase activity chemically or genetically led to LSCC Hep2 cell cycle arrest and apoptotic cell death. Importantly, we found that Aur-A increases cell migration and this novel function was correlated with Akt1 activation. The enhanced cell migration induced by Aur-A overexpression could be abrogated by either small-molecule Akt1 inhibitor or short interfering RNA. VX-680, a selective Aurora kinase inhibitor, decreased Akt1 phosphorylation at Ser(473) and inhibited cell migration, but failed to do so in constitutive active Akt1 (myr-Akt1)-overexpressed cells. Moreover, our data suggested that overexpression of Aur-A kinase might also contribute to radioresistance of LSCC. Inhibiting Aur-A by VX-680 induced expression of p53 and potently sensitized cells to radiotherapy, leading to significant cell death. Ectopic overexpression of Aur-A, however, reduced p53 level and rendered cells more resistant to irradiation. Taken together, we showed that Aur-A kinase, a negative prognostic marker, promotes migration and reduces radiosensitivity in laryngeal cancer cells.

Elevated Beclin 1 expression is correlated with HIF-1α in predicting poor prognosis of nasopharyngeal carcinoma

Recent studies have suggested that autophagy plays a pivotal role in regulation of cancer development and progression. High expression of the autophagy-related Beclin 1 protein predicted favorable patient outcome in several tumors. Here, a randomized controlled trial (RCT)-derived 128 nasopharyngeal carcinoma (NPC) patients were subjected to analysis of Beclin 1 expression and survival probability. In this RCT, 61 patients treated with induction chemotherapy plus concurrent chemoradiotherapy were used as a training set to generate a Beclin 1 cutoff score for patient outcome by receiver operating characteristic (ROC) curve analysis. For validation, the ROC-derived cutoff point was subjected to analysis of the association of Beclin 1 expression with patient outcome and clinical characteristics in testing set. The testing set comprised of 67 patients received induction chemotherapy plus radiotherapy. In the testing set and overall patients, our univariate and multivariate analysis showed that higher Beclin 1 expression, defined by the training set ROC analysis-generated cutoff score, predicted poorer overall survival, progression-free survival and distant metastasis-free survival. However, we failed to detect a correlation between Beclin 1 and local failure-free survival. Moreover, a positive relationship between Beclin 1 and HIF-1α expression was found. Importantly, among patients with elevated HIF-1α expression, a subset with lower Beclin 1 expression displayed a significant overall survival advantage than those with higher expression (p = 0.036). Contrary to previous studies, our results demonstrated that high autophagic Beclin 1 expression was an inferior prognostic marker for NPC. HIF-1α-associated Beclin 1 high expression might facilitate NPC cells surviving from chemoradiotherapy, suggesting a novel therapeutic molecular target for NPC.

Aurora kinase A inhibition-induced autophagy triggers drug resistance in breast cancer cells

We have previously shown that elevated expression of mitotic kinase aurora kinase A (AURKA) in cancer cells promotes the development of metastatic phenotypes and is associated clinically with adverse prognosis. Here, we first revealed a clinically positive correlation between AURKA and autophagy-associated protein SQSTM1 in breast cancer and further demonstrated that AURKA regulated SQSTM1 through autophagy. Indeed, depletion by siRNA or chemical inhibition of AURKA by the small molecule VX-680 increased both the level of microtubule-associated protein 1 light chain 3-II (LC3-II) and the number of autophagosomes, along with decreased SQSTM1. Conversely, overexpression of AURKA inhibited autophagy, as assessed by decreased LC3-II and increased SQSTM1 either upon nutrient deprivation or normal conditions. In addition, phosphorylated forms of both RPS6KB1 and mechanistic target of rapamycin (MTOR) were elevated by overexpression of AURKA whereas they were suppressed by depletion or inhibition of AURKA. Moreover, inhibition of MTOR by PP242, an inhibitor of MTOR complex1/2, abrogated the changes in both LC3-II and SQSTM1 in AURKA-overexpressing BT-549 cells, suggesting that AURKA-suppressed autophagy might be associated with MTOR activation. Lastly, repression of autophagy by depletion of either LC3 or ATG5, sensitized breast cancer cells to VX-680-induced apoptosis. Similar findings were observed in cells treated with the autophagy inhibitors chloroquine (CQ) and bafilomycin A1 (BAF). Our data thus revealed a novel role of AURKA as a negative regulator of autophagy, showing that AURKA inhibition induced autophagy, which may represent a novel mechanism of drug resistance in apoptosis-aimed therapy for breast cancer.

The Mitotic Kinase Aurora-A Induces Mammary Cell Migration and Breast Cancer Metastasis by Activating the Cofilin-F-actin Pathway

The mitotic kinase Aurora-A (Aur-A) is required to form the bipolar spindle and ensure accurate chromosome segregation before cell division. Aur-A dysregulation represents an oncogenic event that promotes tumor formation. Here, we report that Aur-A promotes breast cancer metastasis. Aur-A overexpression enhanced mammary cell migration by dephosphorylation and activation of cofilin, which facilitates actin reorganization and polymerization. Cofilin knockdown impaired Aur-A-driven cell migration and protrusion of the cell membrane. Conversely, overexpression of activated cofilin abrogated the effects of Aur-A knockdown on cell migration. Moreover, Aur-A overexpession increased the expression of the cofilin phosphatase Slingshot-1 (SSH1), contributing to cofilin activation and cell migration. We found that phosphatidylinositol 3-kinase (PI3K) inhibition blocked Aur-A-induced cofilin dephosphorylation, actin reorganization, and cell migration, suggesting crosstalk with PI3K signaling and a potential benefit of PI3K inhibition in tumors with deregulated Aur-A. Additionally, we found an association between Aur-A overexpression and cofilin activity in breast cancer tissues. Our findings indicate that activation of the cofilin-F-actin pathway contributes to tumor cell migration and metastasis enhanced by Aur-A, revealing a novel function for mitotic Aur-A kinase in tumor progression.

Inhibition of Aurora-A suppresses epithelial-mesenchymal transition and invasion by downregulating MAPK in nasopharyngeal carcinoma cells

Mitotic serine/threonine kinase Aurora-A (Aur-A) plays a critical role in regulating centrosome segregation and spindle assemble. Aur-A overexpression causes excessive centrosome duplication and abnormal spindle structure, leading to tumor malignant progression. Here, we investigated Aur-A expression in nasopharyngeal carcinoma (NPC) and the association between Aur-A and NPC invasiveness. We showed that overexpression of Aur-A in tumor tissues was correlated with cranial bone invasion and clinical stage in NPC patients. Suppression of Aur-A by either selective Aurora inhibitory VX-680 or small-interfering RNA caused G(2)/M arrest and apoptotic cell death in NPC CNE-2 cells. Significantly, inhibition of Aur-A suppressed CNE-2 cell invasion and restored membrane expression of epithelial markers, E-cadherin and beta-catenin, suggesting a reversed epithelial-mesenchymal transition process in cancer cells. In addition, we found that Aur-A-regulated epithelial-mesenchymal transition and invasion were mediated by mitogen-activated protein kinase (MAPK) phosphorylation. Moreover, suppression of MAP kinase by small-interfering RNA or its upstream MEK1/2-selective inhibitor U0126 abrogated cell invasion enhanced by Aur-A overexpression. On the other hand, forced overexpression of constitutively active form of MEK1/2, MEK2DD, in CNE-2 cancer cells rescued cell invasive ability suppressed by VX-680-imposed Aur-A inhibition. Our results indicated that Aur-A acted through a downstream MAP kinase pathway to promote epithelial-mesenchymal transition and invasiveness in nasopharyngeal tumorigenesis. Small chemical inhibitor VX-680 may offer as a promising molecular targeting agent in human NPC.

The splicing factor RBM4 controls apoptosis, proliferation, and migration to suppress tumor progression

Splicing dysregulation is one of the molecular hallmarks of cancer. However, the underlying molecular mechanisms remain poorly defined. Here we report that the splicing factor RBM4 suppresses proliferation and migration of various cancer cells by specifically controlling cancer-related splicing. Particularly, RBM4 regulates Bcl-x splicing to induce apoptosis, and coexpression of Bcl-xL partially reverses the RBM4-mediated tumor suppression. Moreover, RBM4 antagonizes an oncogenic splicing factor, SRSF1, to inhibit mTOR activation. Strikingly, RBM4 expression is decreased dramatically in cancer patients, and the RBM4 level correlates positively with improved survival. In addition to providing mechanistic insights of cancer-related splicing dysregulation, this study establishes RBM4 as a tumor suppressor with therapeutic potential and clinical values as a prognostic factor.

High pretreatment serum lactate dehydrogenase level correlates with disease relapse and predicts an inferior outcome in locally advanced nasopharyngeal carcinoma.

PURPOSE Here, we evaluate the prognostic effect of pretreatment serum lactate dehydrogenase (LDH) in locally advanced nasopharyngeal carcinoma (NPC). METHODS AND MATERIALS Pretreatment serum samples from a randomized controlled trial, which contained 199 neoadjuvant chemoradiotherapy patients and 201 neoadjuvant-concurrent chemoradiotherapy cases with locally advanced NPC, were collected and examined for LDH. With 5-year follow-up, the prognostic effect of pretreatment serum LDH was analysed by Kaplan-Meier analysis and multivariate Cox regression model. RESULTS Three hundred and sixty-seven patients (91.75%) had a normal (109.0-245.0 U/L) pretreatment LDH level, compared to 33 cases (8.25%) that had a higher (≥245.0 U/L) LDH level. The mean and median pretreatment LDH levels of these 400 patients were 186.6 and 174.0 U/L (range, 83.0-751.0 U/L), respectively. Compared with the normal subset, elevated LDH level predicted an inferior 5-year overall survival (56.9% versus 76.8%, P=0.004), disease-free survival (DFS, 45.4% versus 64.7%, P=0.001), local relapse-free survival (76.1% versus 89.6%, P=0.019) and distant metastasis-free survival (DMFS, 54.3% versus 72.2%, P=0.001). Multivariate analysis confirmed that the LDH level was an independent prognostic factor to predict death, disease progression, local relapse and distant metastasis. For the subgroup with normal LDH (median point of 177.0 U/L), we detected an evident 5-year DFS (68.8% versus 59.5%, P=0.047) and DMFS advantage (77.3% versus 65.3%, P=0.016) in 109.0-177.0 U/L subset than that of 178.0-245.0 U/L subgroup. CONCLUSIONS Serological LDH level was an independent prognostic factor for locally advanced NPC. Combining pretreatment LDH with TNM staging might lead to more accurate risk definition.

Low expression of Beclin 1, associated with high Bcl-xL, predicts a malignant phenotype and poor prognosis of gastric cancer.

Recent studies have suggested that dysregulation of autophagy plays a pivotal role in tumorigenesis. Here, we determined the prognostic value of autophagy-related protein Beclin 1 in gastric cancer. A total of 153 primary gastric cancer patients were subjected to analysis of Beclin 1 expression and survival prognosis. Among them, 68 patients were assigned randomly and used as a training set to generate a cutoff score for Beclin 1 expression by receive operating characteristic (ROC) curve analysis. The ROC-generated cutoff score was subjected to analyze the association of Beclin 1 with clinical characteristics and patient outcome. In a testing set (n = 85) and overall patients (n = 153), both univariate and multivariate analysis found that low expression of Beclin 1 predicted adverse overall survival and progression-free survival for gastric cancer patients. Furthermore, in each stage of gastric cancer patients, Beclin 1 expression was a prognostic indicator in patients with stage II, III and IV. Importantly, a reverse relationship between Beclin 1 and Bcl-xL expression was demonstrated. In patients of elevated Bcl-xL expression, a subset with lower Beclin 1 expression displayed an inferior overall survival and progression-free survival than those with higher Beclin 1 expression. Thus, our data demonstrated that low expression of Beclin 1, associated with high Bcl-xL, played as an independent biomarker, contributing to a more aggressive cancer cell phenotype and poor prognosis for gastric tumor.