A novel miR-200c/c-myc negative regulatory feedback loop is essential to the EMT process, CSC biology and drug sensitivity in nasopharyngeal cancer.

Overexpression of the c-Myc oncogene has been implicated in cancer stem cell - like (CSC) phenotypes and epithelial-to-mesenchymal transition (EMT) in cancer. However, the underlying molecular mechanism by which c-Myc regulates EMT and CSC potential in remains unclear. In the present study, we showed that the expression of c-Myc protein is inversely correlated with microRNA (miR)-200c expression in primary tumor samples from nasopharyngeal cancer (NPC) patients. We further demonstrated that Myc and miR-200c negatively regulate the expression each other in NPC cell lines. c-Myc transcriptionally repressed expression of miR-200c by directly binding to two E-box sites located within a 1 kb segment upstream of TSS of the miR-200c. In addition, miR-200c post-transcriptionally repressed expression of c-Myc by binding to its 3'-untranslated region, suggesting the existence of a negative feedback loop between Myc and miR-200c. Overexpression of c-Myc interfered with this feedback loop and activated the EMT program, induced CSC phenotypes, and enhanced drug sensitivity, whereas miR-200c could counteract these biological effects of c-Myc. Our results provide a novel mechanism governing c-Myc and miR-200c expression and indicate that either targeting c-Myc or restoring miR-200c expression would be a promising approach to overcome oncogenic role of c-Myc in NPC.

Knockdown of lncRNA ZFAS1-suppressed non-small cell lung cancer progression via targeting the miR-150-5p/HMGA2 signaling.

Non-small cell lung cancer (NSCLC) is the main type of lung malignancy. Early diagnosis and treatments for NSCLC are far from satisfactory due to the limited knowledge of the molecular mechanisms regarding NSCLC progression. Long noncoding RNA (lncRNA) ZNFX1 antisense RNA1 (ZFAS1) has been implicated for its functional role in the progression of malignant tumors. This study aimed to determine the ZFAS1 expression from lung cancer clinical samples and to explore the molecular mechanisms underlying ZFAS1-modulated NSCLC progression. Experimental assays revealed that clinical samples and cell lines of lung malignant tumors showed an upregulation of ZFSA1. ZFAS1 expression was markedly upregulated in the lung tissues from patients with advanced stage of this malignancy. The loss-of-function assays showed that knockdown of ZFAS1-suppressed NSCLC cell proliferative, as well as invasive potentials, increased NSCLC cell apoptotic rates in vitro and also attenuated tumor growth of NSCLC cells in the nude mice. Further experimental evidence showed that ZFAS1 inversely affected miR-150-5p expression and positively affected high-mobility group AT-hook 2 (HMGA2) expression in NSCLC cell lines. MiR-150-5p inhibition or HMGA2 overexpression counteracted the effects of ZFAS1 knockdown on NSCLC cell proliferative, invasive potentials and apoptotic rates. In light of examining the clinical lung cancer samples, miR-150-5p expression was downregulated and the HMGA2 expression was highly expressed in the lung cancer tissues compared with normal ones; the ZFAS1 expression showed a negative correlation with miR-150-5p expression but a positive correlation with HMGA2 expression in lung cancer tissues. To summarize, we, for the first time, demonstrated the inhibitory effects of ZFAS1 knockdown on NSCLC cell progression, and the results from mechanistic studies indicated that ZFAS1-mediated NSCLC progression cells via targeting miR-150-5p/HMGA2 signaling.

Case report: Durable response to alectinib in ALK-rearranged lung adenocarcinoma with acquired, crizotinib-resistant ALK C1156F mutation.

Treatment of ALK-rearranged non-small cell lung cancer (NSCLC) with tyrosine kinase inhibitors (TKIs) is challenged by the almost inevitable emergence of therapeutic resistance. Different profiles of resistance mechanisms have been reported for the currently available ALK TKIs. The ALK C1156Y mutation is reported in 2% of patients with acquired resistance to crizotinib. A rare substitution at the same site, C1156F, remains largely unknown. Existing evidence includes identification of C1156F and G1202R in an alectinib-resistant patient and sensitivity to crizotinib and resistance to later-generation 3ALK inhibitors in preclinical models. In this report, we present two cases in which NSCLC patients acquired the ALK C1156F mutation on crizotinib monotherapy. Both patients were men, and one had been heavily treated with chemotherapeutic regimens before identification of ALK rearrangement, whereas the other received crizotinib as first-line treatment. Genomic profiling of blood biopsies after progression on crizotinib suggested emergence of the ALK C1156F variant. Both patients subsequently received and responded favorably to alectinib, achieving respective progression-free survival of 21 and 15 months as of the latest follow-ups. To the best of our knowledge, this work is the first to provide clinical evidence of resistance to crizotinib and sensitivity to alectinib in NSCLC patients harboring acquired ALK C1156F mutation.

Targeted sequencing identifies the mutational signature of double primary and metastatic malignancies: a case report.

BACKGROUND: The accurate identification of the tissue of origin is critical for optimal management of cancer patients particularly those who develop multiple malignancies; however, conventional diagnostic methods at times may fail to provide conclusive diagnosis of the origin of the malignancy. Herein, we describe the use of targeted sequencing in distinguishing the primary and metastatic tumors in a patient with metachronous malignancies in the lung, colon and kidney. CASE PRESENTATION: In December 2016, a 55-year-old Chinese male was diagnosed with stage IB lung adenosquamous carcinoma and treated with left lower lobectomy and 4 cycles of platinum-based chemotherapy. After being disease-free for 3.5 months, three colonic polyps were discovered and were diagnosed as invasive adenocarcinoma after polypectomy. Within 5.4 months from the polypectomy, squamous cell renal carcinoma was identified and was managed by radical nephrectomy. Immunohistochemistry results were inconclusive on the origin of the kidney tumor. Hence, the three archived surgical tissue samples were sequenced using a targeted panel with 520 cancer-related genes. Analysis revealed similar mutational signature between the lung and kidney tumors and a distinct mutational profile for the colon tumor, suggesting that the lung and colon malignancies were primary tumors, while the kidney tumor originated from the lung, revealing a diagnosis of metastatic double primary cancer - lung carcinoma with renal cell metastasis and second primary colon carcinoma. CONCLUSION: Mutational profiling using targeted sequencing is valuable not only for the detection of actionable mutations, but also in the identification of the origin of tumors. This diagnostic approach should be considered in similar scenarios.

MiR-219a-5p enhances cisplatin sensitivity of human non-small cell lung cancer by targeting FGF9.

Cisplatin (DDP) resistance is a major obstacle in the treatment of non-small cell lung cancer (NSCLC). MicroRNA-219a-5p (miR-219a-5p) has been reported to be a tumor suppressor in several cancers, but whether it regulates chemosensitivity in NSCLC remains unclear. Here, using quantitative real time PCR analysis, we observed that miR-219a-5p was down-regulated in responding tumor tissues compared with that in non-responding tumor tissues from NSCLC patients received DDP-based chemotherapy. Consistently, miR-219a-5p expression was lower in cisplatin (DDP)-resistant NSCLC cell lines (A549-R and SPC-A1-R) than that in corresponding parental cells (A549 and SPC-A1). Gain of-function assay showed ectopic expression of miR-219a-5p reversed DDP chemoresistance of NSCLC cells in vitro and in vivo. Using bioinformatics prediction and dual-luciferase reporter assays, we identified the FGF9 gene as a novel direct target of miR-219a-5p. Moreover, restoration of FGF9 expression reversed the miR-219a-5p-mediated chemosensitivity. In conclusion, this study demonstrated miR-219a-5p plays a crucial role in the development of acquired drug resistance to DDP in NSCLC cells by targeting FGF9 and might be a therapeutic target for DDP resistance in clinical practice.

The clinical characteristics and prognostic analysis of Chinese advanced NSCLC patients based on circulating tumor DNA sequencing.

PURPOSE: Circulating tumor DNA (ctDNA) is a noninvasive and real-time marker for tumor diagnosis, prognosis, and prediction. However, further investigations about ctDNA prognostic and predictive value are still needed, and conclusions from several studies were inconsistent. EXPERIMENTAL DESIGN: We performed capture-based targeted ultradeep sequencing on liquid biopsies from a cohort of 34 advanced Chinese non-small-cell lung cancer (NSCLC) patients and analyzed the clinical use of ctDNA in this study. RESULTS: On the basis of clinical characteristics of the 34 NSCLC patients, we found that brain metastasis correlated with shorter progression-free survival (PFS) and is more prone to happen in younger patients. After ctDNA sequencing, we analyzed the prognostic value of baseline ctDNA. In osimertinib-treated group, high max allelic fraction (maxAF) correlated with shorter PFS. But for the cohort of 34 patients, no correlation can be observed between maxAF and PFS. We also presented two cases to demonstrate the value of disease progression prediction by ctDNA, which can be detected earlier than clinical response. CONCLUSION: In this study, we demonstrated that ctDNA is a prognostic marker for evaluating treatment response and predicting recurrence in advanced NSCLC. Further investigations with larger cohort and uniformed patient background are still needed to validate our findings.

Down-regulation of microRNA-224 -inhibites growth and epithelial-to-mesenchymal transition phenotype -via modulating SUFU expression in bladder cancer cells.

Aberrant expression of miR-224 is usually found in cancer studies; however, the role of miR-224 has seldom been reported in bladder cancer (BC). We explored miR-224's function and the underlying mechanism in BC. It was found that miR-224 expression was significantly up-regulated in BC tissues and cell lines. Knockdown of miR-224 decreased BC cell growth and invasion both in vitro and in vivo. We identified the SUFU protein as a downstream target of miR-224 by using luciferase and western blot assays. We proposed that miR-224 promoted BC cell growth and invasion via sustaining the activity of Hedgehog pathway, which was negatively regulated by SUFU. Taken together, our study demonstrated that miR-224 may function as an onco-miR in BC and suggested that miR-224 may be a potential therapeutic target for BC patients.

LncRNA ZEB2-AS1 promotes pancreatic cancer cell growth and invasion through regulating the miR-204/HMGB1 axis.

Recently, lncRNA ZEB2-AS1 was identified as a lncRNA that promoted cancer progression. However, the biological function and the underlying mechanism of ZEB2-AS1 in pancreatic cancer had not been reported. In the current study, we revealed that the expression level of ZEB2-AS1 was elevated in pancreatic cancer cell lines and tissues. ZEB2-AS1 inhibition decreased cell growth and invasion in pancreatic cancer. Mechanismly, ZEB2-AS1 exerted as a ceRNA and negatively regulated miR-204 expression. In addition, HMGB1 was identified as a down-stream target of miR-204. The miR-204/HMGB1 axis mediated ZEB2-AS1's effect on pancreatic cancer. Our findings revealed that lncRNA ZEB2-AS1 may be a candidate prognostic biomarker and a target for new therapies in pancreatic cancer patients.

Methylation-mediated silencing of microRNA-211 promotes cell growth and epithelial to mesenchymal transition through activation of the AKT/ß-catenin pathway in GBM.

Aberrant expression of miR-211 has frequently been reported in cancer studies; however, its role in glioblastoma multiforme (GBM) has not been examined in detail. We investigated the function and the underlying mechanism of miR-211 in GBM. We revealed that miR-211 was downregulated in GBM tissues and cell lines. Restoration of miR-211 inhibited GBM cell growth and invasion both in vitro and in vivo. The epithelial to mesenchymal transition (EMT) phenotype was reversed when miR-211 expression was restored. HMGA2 was identified as a down-stream target of miR-211. MiR-211 had an inhibitory effect on AKT/ß-catenin signaling, which was reversed by HMGA2 overexpression or miR-211 restoration. In addition, miR-211 was transcriptionally repressed by EZH2-induced H3K27 trimethylation and promoter methylation. Overall, our findings revealed miR-211 as a tumor suppressor in GBM and mir-211 may be a potential therapeutic target for GBM patients.

Sulforaphane inhibits cancer stem-like cell properties and cisplatin resistance through miR-214-mediated downregulation of c-MYC in non-small cell lung cancer.

We herein report that sulforaphane (SFN), a potent anti-cancer and well-tolerated dietary compound, inhibits cancer stem-like cell (CSC) properties and enhances therapeutic efficacy of cisplatin in human non-small cell lung cancer (NSCLC). SFN exerted these functions through upregulation of miR-214, which in turn targets the coding region of c-MYC. This finding was further corroborated by our observations that plasmid or lentiviral vector-mediated expression of 3'UTR-less c-MYC cDNA and cisplatin- or doxorubicin-induced endogenous c-MYC accumulation was similarly suppressed by either SFN or miR-214. Further, we showed that the reported inhibitory effects of SFN on ß-catenin are also mediated by miR-214. SFN/miR-214 signaling inhibited CSC properties and enhanced the cytotoxicity of chemotherapeutic drugs in vitro. Experiments with nude mice carrying xenograft tumors showed that SFN sensitized NSCLC cells to cisplatin's efficacy, which is accompanied by inhibition of cisplatin-induced c-MYC accumulation in tumor tissues. Our results provided strong evidence and mechanisms to support consideration of SFN or synthetic derivatives as a therapeutic agent in combination with cisplatin for the treatment of patients with NSCLC and, potentially, other types of c-MYC-addicted tumors.

BCI Competition 2003--Data set IIb: enhancing P300 wave detection using ICA-based subspace projections for BCI applications.

An algorithm based on independent component analysis (ICA) is introduced for P300 detection. After ICA decomposition, P300-related independent components are selected according to the a priori knowledge of P300 spatio-temporal pattern, and clear P300 peak is reconstructed by back projection of ICA. Applied to the dataset IIb of BCI Competition 2003, the algorithm achieved an accuracy of 100% in P300 detection within five repetitions.

Cancer stem-like cell properties are regulated by EGFR/AKT/ß-catenin signaling and preferentially inhibited by gefitinib in nasopharyngeal carcinoma.

We report that the epidermal growth factor receptor (EGFR) pathway plays a critical role in regulating cancer stem-like cells (CSCs) in nasopharyngeal carcinoma (NPC), one of the most common malignant tumors in Southeast Asia. Effects of EGFR on maintaining CSCs are mainly mediated by AKT signaling, and ß-catenin is responsible for governing CSC properties in response to EGFR/AKT activation. Significantly, CSCs are enriched by cisplatin and decreased by gefitinib in NPC xenograft models. Upon reimplantation in secondary mice, tumor cells derived from cisplatin-treated mice grew rapidly, whereas regrowth of tumor cells from gefitinib-treated mice was severely diminished. We further demonstrate that expression of EGFR correlates with expression of ß-catenin and Nanog in primary tumor specimens from NPC patients. These findings provide mechanistic and preclinical evidence supporting the use of gefitinib alone or in combination with a chemotherapeutic agent in first-line therapy for patients with NPC. In addition, our results suggest that targeting ß-catenin represents a rational clinical modality for patients whose tumors harbor activated EGFR or AKT.

Esophageal cancer tumorspheres involve cancer stem-like populations with elevated aldehyde dehydrogenase enzymatic activity.

Cancer stem cells (CSCs) form spheres in vitro in serum-free suspension culture. Sphere formation is particularly useful to enrich the potential CSC subpopulations as a functional approach. Few reports are currently available on tumorspheres in esophageal cancer (EC). The present study focused on evaluating the cancer stem-like properties and analyzing the difference between spheroid and adherent cells of the Eca109 human EC cell line. Immunofluorescence and immunoblotting analysis revealed that EC tumorspheres expressed the stem cell markers Nanog and Oct4 more highly, but showed a decreased expression of the differentiation marker CK5/6. The spheroids were chemoresistant to cisplatin compared to the adherent cells (32.5 vs. 135.8 µM in IC50). Side population cells increased in tumorspheres compared to adherent cells (0.7 vs. 5.6%). A marked upregulation of drug-resistant genes (ABCG2 and MDR1) was observed in sphere-forming cells. We compared the profiles of adherent and spheroid cells by microarrays and obtained one representative differentially expressed gene, aldehyde dehydrogenase (ALDH). We also verified that the cancer stem-like cells of EC contained a high ALDH enzymatic activity. ALDH-positive cells were enriched by 11- to 12-fold in spheroids, compared to adherent cells (2.5 vs. 28.6%). Immunofluorescence and immunoblotting analysis also revealed a higher expression of ALDH in EC tumorspheres. In conclusion, our study verified that sphere-forming culturing can be utilized to demonstrate the putative esophageal CSCs, and identified a potential esophageal CSC surface marker, ALDH.

Dynamic EEG Analysis via the Variability of Band Relative Intensity Ratio: A Time-Frequency Method.

The effectiveness of band relative intensity ratio (BRIR) based on Time-Frequency Representations (TFRS) was explored as a method to depict brain dynamics in this paper. Electroencephalographic (EEG) signals from 20 subjects through 16 channels have been studied under open eyes state and closed eyes state. The results indicate that EEG rhythm features and the dynamic process of brain activity can be revealed explicitly though the time-frequency variability and the time-frequency- space variability of BRIR. The alpha rhythm is best seen under eyes closed state and blocked when eyes are open generally, but it is strengthened in some times when it is blocked overall under open eyes state.