R-loop resolution by ARIP4 helicase promotes androgen-mediated transcription induction.

Pausing of RNA polymerase II (Pol II) at transcription start sites (TSSs) primes target genes for productive elongation. Coincidentally, DNA double-strand breaks (DSBs) enrich at highly transcribed and Pol II-paused genes, although their interplay remains undefined. Using androgen receptor (AR) signaling as a model, we have uncovered AR-interacting protein 4 (ARIP4) helicase as a driver of androgen-dependent transcription induction. Chromatin immunoprecipitation sequencing analysis revealed that ARIP4 preferentially co-occupies TSSs with paused Pol II. Moreover, we found that ARIP4 complexes with topoisomerase II beta and mediates transient DSB formation upon hormone stimulation. Accordingly, ARIP4 deficiency compromised release of paused Pol II and resulted in R-loop accumulation at a panel of highly transcribed AR target genes. Last, we showed that ARIP4 binds and unwinds R-loops in vitro and that its expression positively correlates with prostate cancer progression. We propose that androgen stimulation triggers ARIP4-mediated unwinding of R-loops at TSSs, enforcing Pol II pause release to effectively drive an androgen-dependent expression program.

Penaeid shrimp counteract high ammonia stress by generating and using functional peptides from hemocyanin, such as HMCs27.

Agricultural and anthropogenic activities release high ammonia levels into aquatic ecosystems, severely affecting aquatic organisms. Penaeid shrimp can survive high ammonia stress conditions, but the underlying molecular mechanisms are unknown. Here, total hemocyanin and oxyhemocyanin levels decreased in Penaeus vannamei plasma under high ammonia stress. When shrimp were subjected to high ammonia stress for 12 h, 24 hemocyanin (HMC) derived peptides were identified in shrimp plasma, among which one peptide, designated as HMCs27, was chosen for further analysis. Shrimp survival was significantly enhanced after treatment with the recombinant protein of HMCs27 (rHMCs27), followed by high ammonia stress. Transcriptome analysis of shrimp hepatopancreas after treatment with or without rHMCs27 followed by high ammonia stress revealed 973 significantly dysregulated genes, notable among which were genes involved in oxidation and metabolism, such as cytochrome C, catalase (CAT), isocitrate dehydrogenase, superoxide dismutase (SOD), trypsin, chymotrypsin, glutathione peroxidase, glutathione s-transferase (GST), and alanine aminotransferase (ALT). In addition, levels of key biochemical indicators, such as SOD, CAT, and total antioxidant capacity (T-AOC), were significantly enhanced, whereas hepatopancreas malondialdehyde levels and plasma pH, NH3, GST, and ALT levels were significantly decreased after rHMCs27 treatment followed by high ammonia stress. Moreover, high ammonia stress induced hepatopancreas tissue injury and apoptosis, but rHMCs27 treatment ameliorated these effects. Collectively, the current study revealed that in response to high ammonia stress, shrimp generate functional peptides, such as peptide HMCs27 from hemocyanin, which helps to attenuate the ammonia toxicity by enhancing the antioxidant system and the tricarboxylic acid cycle to decrease plasma NH3 levels and pH.

The histidine phosphatase LHPP of Penaeus vannamei is involved in shrimp hemocytes apoptosis.

LHPP (Phospholysine Phosphohistidine Inorganic Pyrophosphate Phosphatase) is a protein histidine phosphatase that modulates a hidden posttranslational modification called histidine phosphorylation. LHPP also acts as a tumor suppressor, which plays a pivotal role in various cellular processes. However, whether LHPP participates in the regulation of invertebrate's immunity is still unknown. Here we characterized a LHPP homolog in P. vannamei (designated PvLHPP), with a 807 bp length of open reading frame (ORF) encoding a putative protein of 268 amino acids. Sequence analysis revealed that PvLHPP contains a typical hydrolase 6 and hydrolase-like domain, which was conserved from invertebrate to vertebrate. PvLHPP was ubiquitously expressed in tissues and induced in hemocyte and hepatopancreas by Vibrio parahaemolyticus, Streptococcus iniae and white spot syndrome virus (WSSV) challenge, indicating that PvLHPP participated in the immune responses. Moreover, silencing of PvLHPP followed by V. parahaemolyticus inhibited hemocyte apoptosis. This study enriches our current insight on shrimp immunity, and provides novel perspective to understand immune-regulatory role of PvLHPP.

A transcriptomic map of EGFR-induced epithelial-to-mesenchymal transition identifies prognostic and therapeutic targets for head and neck cancer.

BACKGROUND: Epidermal growth factor receptor (EGFR) is both a driver oncogene and a therapeutic target in advanced head and neck squamous cell carcinoma (HNSCC). However, response to EGFR treatment is inconsistent and lacks markers for treatment prediction. This study investigated EGFR-induced epithelial-to-mesenchymal transition (EMT) as a central parameter in tumor progression and identified novel prognostic and therapeutic targets, and a candidate predictive marker for EGFR therapy response. METHODS: Transcriptomic profiles were analyzed by RNA sequencing (RNA-seq) following EGFR-mediated EMT in responsive human HNSCC cell lines. Exclusive genes were extracted via differentially expressed genes (DEGs) and a risk score was determined through forward feature selection and Cox regression models in HNSCC cohorts. Functional characterization of selected prognostic genes was conducted in 2D and 3D cellular models, and findings were validated by immunohistochemistry in primary HNSCC. RESULTS: An EGFR-mediated EMT gene signature composed of n = 171 genes was identified in responsive cell lines and transferred to the TCGA-HNSCC cohort. A 5-gene risk score comprising DDIT4, FADD, ITGB4, NCEH1, and TIMP1 prognosticated overall survival (OS) in TCGA and was confirmed in independent HNSCC cohorts. The EGFR-mediated EMT signature was distinct from EMT hallmark and partial EMT (pEMT) meta-programs with a differing enrichment pattern in single malignant cells. Molecular characterization showed that ITGB4 was upregulated in primary tumors and metastases compared to normal mucosa and correlated with EGFR/MAPK activity in tumor bulk and single malignant cells. Preferential localization of ITGB4 together with its ligand laminin 5 at tumor-stroma interfaces correlated with increased tumor budding in primary HNSCC tissue sections. In vitro, ITGB4 knock-down reduced EGFR-mediated migration and invasion and ITGB4-antagonizing antibody ASC8 impaired 2D and 3D invasion. Furthermore, a logistic regression model defined ITGB4 as a predictive marker of progression-free survival in response to Cetuximab in recurrent metastatic HNSCC patients. CONCLUSIONS: EGFR-mediated EMT conveyed through MAPK activation contributes to HNSCC progression upon induction of migration and invasion. A 5-gene risk score based on a novel EGFR-mediated EMT signature prognosticated survival of HNSCC patients and determined ITGB4 as potential therapeutic and predictive target in patients with strong EGFR-mediated EMT.

Prognostic models for 1-year survival of NPC after radiotherapy in different ages.

PURPOSE: Previous studies have shown that approximately 10% of nasopharyngeal cancer (NPC) patients die within a year of disease onset, and that age is an independent predictor. However, no predictive model has been developed. We aimed to establish novel prognostic models to predict the 1-year cancer-specific survival (CSS) of young, middle-aged, and older patients with NPC after radiotherapy. METHODS: The data of 2822 NPC patients who underwent radiotherapy between 2004 and 2015 were reviewed from the surveillance, epidemiology, and end results database. We divided them into young, middle-aged, and older people groups according to age (< 44 years, 45-59 years, and ≥ 60 years, respectively). Multivariate analyses were performed, and prognostic models were constructed. RESULTS: Multivariate analyses indicated that age, ethnicity, histological subtype, T, and M stage were independent predictors of 1-year CSS in the older people group. In contrast, ethnicity and age were not found to have predictive value in the young and middle-aged groups, respectively. Accordingly, three prognostic models with excellent predictive values were established for the three groups (C-indices: 0.791 [95% CI 0.722-0.859], 0.763 [95% CI 0.721-0.806] and 0.723 [95% CI 0.683-0.763], respectively). These predictive values are higher than those of the eighth edition American joint committee cancer tumor-node-metastasis (TNM) classification system. CONCLUSION: Three prognostic models for predicting the 1-year CSS of young, middle-aged, and older NPC patients after radiotherapy showed better predictive power than the TNM classification system. These models may guide treatment strategies and clinical decision-making in different cohorts.

Long-term survival trend after primary total laryngectomy for patients with locally advanced laryngeal carcinoma.

Purpose: To evaluate long-term survival trends after primary total laryngectomy (TL) for locally advanced laryngeal carcinoma (LC). Methods: A total of 2094 patients diagnosed with locally advanced LC and underwent primary TL (1992-2011, at least 5-year follow-up) in the Surveillance, Epidemiology, and End Results (SEER) database were included in this study. Besides the traditional overall survival (OS) and cancer-specific survival (CSS) by using Kaplan-Meier curves, the 3-year conditional survival analysis was also performed to describe the long-term trends in these patients. Time-dependent multivariate competing-risk models were constructed to assess the persistent sub-distribution hazard of prognostic factors. Finally, a nomogram was developed to predict conditional cancer-specific survival. Results: The curves of overall hazard and cancer-specific hazard both quickly reached the apex within the first year since TL, then decreased thereafter. In general, the CS3 steadily increased from within 5 years after TL. In the stratified CS3 analysis, the increments in patients with adverse characteristics were more pronounced. 4 years after TL, the probability of surviving the next year exceeded 90%. The time-dependent multivariate competing-risk models indicated that age and lymph node ratio (LNR) persistently contributed to the cancer-specific outcome. The nomogram based on the competing-risk model was constructed to estimate CSS probability conditional upon 3 years for advanced LC patients having survived 1, 2, and 3 years. Conclusion: Most patients achieved a substantially improved survival rate after surviving a long period after primary TL. Patients diagnosed at older age and with higher LNR should receive more effective follow-up. The predictive nomogram can provide significant evidence for clinical research and practice.

The primary tumor resection in patients with distant metastatic laryngeal carcinoma.

BACKGROUND: The role of primary tumor resection in patients with distant metastatic laryngeal carcinoma (DMLC) has not been clarified completely. Thus, we used propensity score matching (PSM) and survival analysis to address this issue. METHODS: The PSM was utilized to avoid selection bias and disproportionate distributions of the confounding factors. Kaplan-Meier estimates and Cox proportional hazard analysis were utilized to evaluate overall survival (OS) and cancer-specific survival (CSS). RESULTS: From the Surveillance, Epidemiology, and End Results Program database, a cohort of 480 patients with DMLC were included. After PSM, the OS and CSS for patients who underwent resection were significantly longer than those without resection (median OS: 19 months vs. 8 months, P < 0.001; median CSS: 19 months vs. 9 months, P = 0.002). Tumor resection significantly prolonged survival of DMLC patients with appropriate demographic and clinical characteristics. In the multivariate analysis, age at diagnosis, race, pathologic subtype, and marital status were found significantly affecting both OS and CSS of patients who underwent surgical resection. Predictive nomograms were developed to help distinguish patients with early mortality potential after surgical resection. CONCLUSIONS: This study is the first one using PSM to assess the role played by surgical resection in DMLC and evaluate the prognostic factor of resected patients. Premised on well controlled postoperative complications, resection could significantly prolong OS and CSS of certain patients.

Dynamic prediction of cancer-specific survival for primary hypopharyngeal squamous cell carcinoma.

OBJECTIVES: This study investigated a large cohort of patients to construct a predictive nomogram and a web-based survival rate calculator for dynamically predicting the cancer-specific survival of patients with primary hypopharyngeal squamous cell carcinoma (HSCC). METHODS: Patients (n = 2007) initially diagnosed with primary HSCC from 2004 to 2015 were extracted from the Surveillance, Epidemiology, and End Results (SEER) database. All patients were randomly divided into the training and validation cohorts (1:1). The Lasso Cox regression model was applied to identify independent risk factors of cancer-specific survival for a predictive nomogram and a web-based calculator. The model was evaluated by concordance index, calibration, and decision curve analysis. RESULTS: Cancer-specific survival rates decreased with time, while 3-year conditional survival increased. Cancer-specific deaths evolved from relatively high within the first 3 years to low thereafter. Age, race, T stage, N stage, M stage, surgery, radiotherapy, chemotherapy, and marital status were identified as independent risk factors. We constructed a predictive nomogram for survival and a web-based calculator ( https://linzhongyang.shinyapps.io/Hypopharyngeal/ ). Additionally, a prognostic risk stratification was developed according to nomogram total points. CONCLUSIONS: Patients with primary HSCC were found at a high risk of cancer-specific death during the first 3 years, indicating that additional effective follow-up strategies should be implemented over the period. This is the first study to construct a predictive nomogram and a web-based calculator for all patients with HSCC.

Cloning and characterisation of the SpToll gene from green mud crab, Scylla paramamosain.

Toll/Toll-like receptors (TLRs), one of the most important pattern recognition receptors (PRRs), play a crucial role in innate immune responses in both invertebrates and vertebrates. In this study, we cloned and characterised a Toll gene from Scylla paramamosain (SpToll). Bioinformatic analysis predicted that SpToll contained one open reading frame of 3018bp and encoded a single-pass transmembrane domain protein of 1005 amino acids. Further, SpToll could be clustered into one branch along with other arthropod Tolls in a phylogenetic tree. SpToll transcripts could be detected by RT-PCR from all tissues examined including the heart, gill, hepatopancreas, stomach, intestine, muscle, eyestalk and hemocytes. Infection by Vibrio parahemolyticus up-regulated SpToll mRNA expression in hemocytes after 48h. The profile of single nucleotide polymorphisms (SNPs) in the leucine-rich repeats (LRRs) domain of SpToll in three healthy crabs was then evaluated. Two hundred and twenty SNPs with a frequency of about 1.0-4.0% were identified in hemocyte DNA/cDNA. Surprisingly, the adenine to guanine transition at position 1372 (c.1372A>G) had a frequency of about 50%. Finally, the results showed that challenge with V. parahemolyticus stimulated the appearance of two sets of SNPs in crabs. More importantly, the c.1372A>G mutation could contribute to a low mortality after V. parahemolyticus infection and introduce variation of charge and secondary structure into the SpToll polypeptide. In summary, these studies suggested a novel Toll homologue in crab and identified a SNP with potential pathogen-resistant activities. The result will be important for the investigation of crab immune defense mechanisms.