Single-Cell Analyses Reveal Mechanisms of Cancer Stem Cell Maintenance and Epithelial-Mesenchymal Transition in Recurrent Bladder Cancer.

PURPOSE: Bladder cancer treatment remains a major clinical challenge due to therapy resistance and a high recurrence rate. Profiling intratumor heterogeneity can reveal the molecular mechanism of bladder cancer recurrence. EXPERIMENTAL DESIGN: Here, we performed single-cell RNA sequencing and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) on tumors from 13 patients with low recurrence risk, high recurrence risk, and recurrent bladder cancer. RESULTS: Our study generated a comprehensive cancer-cell atlas consisting of 54,971 single cells and identified distinct cell subpopulations. We found that the cancer stem-cell subpopulation is enriched during bladder cancer recurrence with elevated expression of EZH2. We further defined a subpopulation-specific molecular mechanism whereby EZH2 maintains H3K27me3-mediated repression of the NCAM1 gene, thereby inactivating the cell invasive and stemness transcriptional program. Furthermore, taking advantage of this large single-cell dataset, we elucidated the spectrum of epithelial-mesenchymal transition (EMT) in clinical samples and revealed distinct EMT features associated with bladder cancer subtypes. We identified that TCF7 promotes EMT in corroboration with single-cell ATAC with high-throughput sequencing (scATAC-seq) analysis. Additionally, we constructed regulatory networks specific to recurrent bladder cancer. CONCLUSIONS: Our study and analytic approaches herein provide a rich resource for the further study of cancer stem cells and EMT in the bladder cancer research field.

Induction chemotherapy with lobaplatin and fluorouracil versus cisplatin and fluorouracil followed by chemoradiotherapy in patients with stage III-IVB nasopharyngeal carcinoma: an open-label, non-inferiority, randomised, controlled, phase 3 trial.

BACKGROUND: Cisplatin-based induction chemotherapy plus concurrent chemoradiotherapy in the treatment of patients with locoregionally advanced nasopharyngeal carcinoma has been recommended in the National Comprehensive Cancer Network Guidelines. However, cisplatin is associated with poor patient compliance and has notable side-effects. Lobaplatin, a third-generation platinum drug, has shown promising antitumour activity against several malignancies with less toxicity. In this study, we aimed to evaluate the efficacy of lobaplatin-based induction chemotherapy plus concurrent chemoradiotherapy over a cisplatin-based regimen in patients with locoregional, advanced nasopharyngeal carcinoma. METHODS: In this open-label, non-inferiority, randomised, controlled, phase 3 trial done at five hospitals in China, patients aged 18-60 years with previously untreated, non-keratinising stage III-IVB nasopharyngeal carcinoma; Karnofsky performance-status score of at least 70; and adequate haematological, renal, and hepatic function were randomly assigned (1:1) to receive intravenously either lobaplatin-based (lobaplatin 30 mg/m2 on days 1 and 22, and fluorouracil 800 mg/m2 on days 1-5 and 22-26 for two cycles) or cisplatin-based (cisplatin 100 mg/m2 on days 1 and 22, and fluorouracil 800 mg/m2 on days 1-5 and 22-26 for two cycles) induction chemotherapy, followed by concurrent lobaplatin-based (two cycles of intravenous lobaplatin 30 mg/m2 every 3 weeks plus intensity-modulated radiotherapy) or cisplatin-based (two cycles of intravenous cisplatin 100 mg/m2 every 3 weeks plus intensity-modulated radiotherapy) chemoradiotherapy. Total radiation doses of 68-70 Gy (for the sum of the volumes of the primary tumour and enlarged retropharyngeal nodes), 62-68 Gy (for the volume of clinically involved gross cervical lymph nodes), 60 Gy (for the high-risk target volume), and 54 Gy (for the low-risk target volume), were administered in 30-32 fractions, 5 days per week. Randomisation was done centrally at the clinical trial centre of Sun Yat-sen University Cancer Centre by means of computer-generated random number allocation with a block design (block size of four) stratified according to disease stage and treatment centre. Treatment assignment was known to both clinicians and patients. The primary endpoint was 5-year progression-free survival, analysed in both the intention-to-treat and per-protocol populations. If the upper limit of the 95% CI for the difference in 5-year progression-free survival between the lobaplatin-based and cisplatin-based groups did not exceed 10%, non-inferiority was met. Adverse events were analysed in all patients who received at least one cycle of induction chemotherapy. This trial is registered with the Chinese Clinical Trial Registry, ChiCTR-TRC-13003285 and is closed. FINDINGS: From June 7, 2013, to June 16, 2015, 515 patients were assessed for eligibility and 502 patients were enrolled: 252 were randomly assigned to the lobaplatin-based group and 250 to the cisplatin-based group. After a median follow-up of 75·3 months (IQR 69·9-81·1) in the intention-to-treat population, 5-year progression-free survival was 75·0% (95% CI 69·7-80·3) in the lobaplatin-based group and 75·5% (70·0 to 81·0) in the cisplatin-based group (hazard ratio [HR] 0·98, 95% CI 0·69-1·39; log-rank p=0·92), with a difference of 0·5% (95% CI -7·1 to 8·1; pnon-inferiority=0·0070). In the per-protocol population, the 5-year progression-free survival was 74·8% (95% CI 69·3 to 80·3) in the lobaplatin-based group and 76·4% (70·9 to 81·9) in the cisplatin-based group (HR 1·04, 95% CI 0·73 to 1·49; log-rank p=0·83), with a difference of 1·6% (-6·1 to 9·3; pnon-inferiority=0·016). 63 (25%) of 252 patients in the lobaplatin-based group and 63 (25%) of 250 patients in the cisplatin-based group had a progression-free survival event in the intention-to-treat population; 62 (25%) of 246 patients in the lobaplatin-based group and 58 (25%) of 237 patients in the cisplatin-based group had a progression-free survival event in the per-protocol population. The most common grade 3-4 adverse events were mucositis (102 [41%] of 252 in the lobaplatin-based group vs 99 [40%] of 249 in the cisplatin-based group), leucopenia (39 [16%] vs 56 [23%]), and neutropenia (25 [10%] vs 59 [24%]). No treatment-related deaths were reported. INTERPRETATION: Lobaplatin-based induction chemotherapy plus concurrent chemoradiotherapy resulted in non-inferior survival and fewer toxic effects than cisplatin-based therapy. The results of our trial indicate that lobaplatin-based induction chemotherapy plus concurrent chemoradiotherapy might be a promising alternative regimen to cisplatin-based treatment in patients with locoregional, advanced nasopharyngeal carcinoma. FUNDING: National Science and Technology Pillar Program, International Cooperation Project of Science and Technology Program of Guangdong Province, Planned Science and Technology Project of Guangdong Province, and Cultivation Foundation for the Junior Teachers at Sun Yat-sen University. TRANSLATION: For the Chinese translation of the abstract see Supplementary Materials section.

Antioxidants suppress radiation-induced apoptosis via inhibiting MAPK pathway in nasopharyngeal carcinoma cells.

Nasopharyngeal carcinoma (NPC) is relatively sensitive to ionizing radiation, and radiotherapy is the main treatment modality for non-metastatic NPC. Radiation therapy generates overproduction of reactive oxygen species (ROS), which can cause DNA damage and induce apoptosis in tumors, thereby killing the malignant cells. Although dietary antioxidant supplementation reduces oxidative stress and promotes tumor progression, the effects of antioxidants on the NPC cells upon radiation have not been reported. In the present study, we showed that antioxidants (ß-Carotene, NAC, GSH) played an anti-apoptotic role in response to radiation via decreasing ROS production and inhibiting MAPK pathway in NPC cells. Based on that, we conclude that the use of supplemental antioxidants during radiotherapy should be avoided because of the possibility of tumor protection and reduced treatment efficacy.

Neoadjuvant chemotherapy followed by concurrent chemoradiotherapy versus concurrent chemoradiotherapy alone in locoregionally advanced nasopharyngeal carcinoma: A phase III multicentre randomised controlled trial.

BACKGROUND: The role of neoadjuvant chemotherapy (NACT) for locoregionally advanced nasopharyngeal carcinoma (NPC) is unclear. We aimed to evaluate the feasibility and efficacy of NACT followed by concurrent chemoradiotherapy (CCRT) versus CCRT alone in locoregionally advanced NPC. METHODS: Patients with stage III-IVB (excluding T3N0-1) NPC were randomly assigned to receive NACT followed by CCRT (investigational arm) or CCRT alone (control arm). Both arms were treated with 80 mg/m2 cisplatin every 3 weeks concurrently with radiotherapy. The investigational arm received cisplatin (80 mg/m2 d1) and fluorouracil (800 mg/m2 civ d1-5) every 3 weeks for two cycles before CCRT. The primary end-point was disease-free survival (DFS) and distant metastasis-free survival (DMFS). Secondary end-point was overall survival (OS). Survival curves for the time-to-event endpoints were analyzed by the Kaplan-Meier method and compared using the log-rank test. The P value was calculated using the 5-year endpoints. RESULTS: Four hundred seventy six patients were randomly assigned to the investigational (n = 238) and control arms (n = 238). The investigational arm achieved higher 3-year DFS rate (82.0%, 95% CI = 0.77-0.87) than the control arm (74.1%, 95% CI = 0.68-0.80, P = 0.028). The 3-year DMFS rate was 86.0% for the investigational arm versus 82.0% for the control arm, with marginal statistical significance (P = 0.056). However, there were no statistically significant differences in OS or locoregional relapse-free survival (LRRFS) rates between two arms (OS: 88.2% versus 88.5%, P = 0.815; LRRFS: 94.3% versus 90.8%, P = 0.430). The most common grade 3-4 toxicity during NACT was neutropenia (16.0%). During CCRT, the investigational arm experienced statistically significantly more grade 3-4 toxicities (P < 0.001). CONCLUSION: NACT improved tumour control compared with CCRT alone in locoregionally advanced NPC, particularly at distant sites. However, there was no early gain in OS. Longer follow-up is needed to determine the eventual therapeutic efficacy.

Advanced-Stage Nasopharyngeal Carcinoma: Restaging System after Neoadjuvant Chemotherapy on the Basis of MR Imaging Determines Survival.

Purpose To evaluate the prognostic value of the restaging system after neoadjuvant chemotherapy (NACT) in patients with advanced-stage nasopharyngeal carcinoma (NPC). Materials and Methods This study was approved by the clinical research committee and a written informed consent was required before enrolling in the study. Prospectively enrolled were 412 consecutive patients with stage III-IVb NPC treated with NACT followed by concurrent chemotherapy and radiation therapy. Patients were staged before NACT and restaged after NACT. The progression-free survival (PFS) and distant metastasis-free survival (DMFS) were calculated with the Kaplan-Meier method, and differences were compared by using the log-rank test. Results Post-NACT T classification (PFS, P = .001) and N classification (PFS, P < .001; DMFS, P = .001) resulted in better survival curve separations than pre-NACT T classification and N classification. Patients downstaged from N2-N3 to N0-N1 disease had a better prognosis than did patients who continued to have N2-N3 diseases (3-year PFS, 83.8% vs 66.6%, P = .001; 3-year DMFS, 88.0% vs 78.4%, P = .026). Multivariate analysis revealed that post-NACT T classification (hazard ratio [HR] = 1.67; 95% confidence interval [CI]: 1.18, 2.36; P = .003) and post-NACT N classification (HR = 1.54; 95% CI: 1.17, 2.03; P = .002) were independent prognostic factors for PFS; also, post-NACT N classification (HR = 1.48; 95% CI: 1.05, 2.07; P = .025) was an independent prognostic factor for DMFS. Multivariate analysis in patients with N2-N3 disease demonstrated that the N downstaging effects of NACT was the only independent prognostic factor for PFS (HR = 0.48; 95% CI: 0.29, 0.81; P = .006) and DMFS (HR = 0.52; 95% CI: 0.28, 0.97; P = .039). Conclusion The post-NACT stage is more representative of prognosis than the pre-NACT stage in advanced-stage NPC patients, which suggests that major clinical decisions should be based on the post-NACT stage. © RSNA, 2016 Online supplemental material is available for this article.

Identification of N-phenyl-2-(N-phenylphenylsulfonamido)acetamides as new RORγ inverse agonists: Virtual screening, structure-based optimization, and biological evaluation.

Retinoic acid receptor-related orphan receptors (RORs) are ligand-dependent transcriptional factors and members of the nuclear receptor superfamily. RORs regulate inflammation, metabolic disorders and circadian rhythm. RORγ is a promising therapeutic drug target for treating Th17-mediated autoimmune diseases. In our study, we performed structure-based virtual screening and ligand-based virtual screening targeting the RORγ ligand-binding domain and successfully identified N-phenyl-2-(N-phenylphenylsulfonamido) acetamides as a type of RORγ inverse agonist. Among the 28 purchased compounds, C11 was confirmed to be active with micromolar IC50 values in both an AlphaScreen assay (62.58 µM) and a cell-based reporter gene assay (4.54 µM). Structure-guided optimization of the compound C11 led to the identification of compound 39, which significantly enhanced RORγ inhibition with an IC50 value of 630 nM. The RORγ antagonism of 39 was 7-fold higher than that of hit compound C11. These results represent a promising starting point for developing potent small molecule RORγ inverse agonists for the treatment of autoimmune diseases, such as rheumatoid arthritis, psoriasis, and multiple sclerosis.

COX-2 promotes metastasis in nasopharyngeal carcinoma by mediating interactions between cancer cells and myeloid-derived suppressor cells.

The expansion of myeloid-derived suppressor cells (MDSCs) is a common feature of cancer, but its biological roles and molecular mechanism remain unclear. Here, we investigated a molecular link between MDSC expansion and tumor cell metastasis in nasopharyngeal carcinoma (NPC). We demonstrated that MDSCs expanded and were positively correlated with the elevated tumor COX-2 expression and serum IL-6 levels in NPC patients. Importantly, COX-2 and MDSCs were poor predictors of patient disease-free survival (DFS). Knocking down tumor COX-2 expression hampered functional TW03-mediated-MDSC cell (T-MDSC) induction with IL-6 blocking. We identified that T-MDSCs promoted NPC cell migration and invasion by triggering the epithelial-mesenchymal transition (EMT) on cell-to-cell contact, and T-MDSCs enhanced tumor experimental lung metastasis in vivo. Interestingly, the contact between T-MDSCs and NPC cells enhanced tumor COX-2 expression, which subsequently activated the ß-catenin/TCF4 pathway, resulting in EMT of the cancer cells. Blocking transforming growth factor ß (TGFß) or inducible nitric oxide synthase (iNOS) significantly abolished the T-MDSC-induced upregulation of COX-2 and EMT scores in NPC cells, whereas the administration of TGFß or L-arginine supplements upregulated COX-2 expression and EMT scores in NPC cells. These findings reveal that COX-2 is a key factor mediating the interaction between MDSCs and tumor cells, suggesting that the inhibition of COX-2 or MDSCs has the potential to suppress NPC metastasis.

Is nasopharyngeal cancer really a "Cantonese cancer"?

Nasopharyngeal cancer (NPC) is endemic in Southern China, with Guandong province and Hong Kong reporting some of the highest incidences in the world. The journal Science has called it a "Cantonese cancer". We propose that in fact NPC is a cancer that originated in the Bai Yue ("proto Tai Kadai" or "proto Austronesian" or "proto Zhuang") peoples and was transmitted to the Han Chinese in southern China through intermarriage. However, the work by John Ho raised the profile of NPC, and because of the high incidence of NPC in Hong Kong and Guangzhou, NPC became known as a Cantonese cancer. We searched historical articles, articles cited in PubMed, Google, monographs, books and Internet articles relating to genetics of the peoples with high populations of NPC. The migration history of these various peoples was extensively researched, and where possible, their genetic fingerprint identified to corroborate with historical accounts. Genetic and anthropological evidence suggest there are a lot of similarities between the Bai Yue and the aboriginal peoples of Borneo and Northeast India; between Inuit of Greenland, Austronesian Mayalo Polynesians of Southeast Asia and Polynesians of Oceania, suggesting some common ancestry. Genetic studies also suggest the present Cantonese, Minnans and Hakkas are probably an admixture of northern Han and southern Bai Yue. All these populations have a high incidence of NPC. Very early contact between southern Chinese and peoples of East Africa and Arabia can also account for the intermediate incidence of NPC in these regions.