A druggable cascade links methionine metabolism to epigenomic reprogramming in squamous cell carcinoma.

Upper aerodigestive squamous cell carcinoma (UASCC) is a common and aggressive malignancy with few effective therapeutic options. Here, we investigate amino acid metabolism in this cancer, surprisingly noting that UASCC exhibits the highest methionine level across all human cancers, driven by its transporter LAT1. We show that LAT1 is also expressed at the highest level in UASCC, transcriptionally activated by UASCC-specific promoter and enhancers, which are directly coregulated by SCC master regulators TP63/KLF5/SREBF1. Unexpectedly, unbiased bioinformatic screen identifies EZH2 as the most significant target downstream of the LAT1-methionine pathway, directly linking methionine metabolism to epigenomic reprogramming. Importantly, this cascade is indispensable for the survival and proliferation of UASCC patient-derived tumor organoids. In addition, LAT1 expression is closely associated with cellular sensitivity to inhibition of the LAT1-methionine-EZH2 axis. Notably, this unique LAT1-methionine-EZH2 cascade can be targeted effectively by either pharmacological approaches or dietary intervention in vivo. In summary, this work maps a unique mechanistic cross talk between epigenomic reprogramming with methionine metabolism, establishes its biological significance in the biology of UASCC, and identifies a unique tumor-specific vulnerability which can be exploited both pharmacologically and dietarily.

Splicing factor hnRNPA1 regulates alternative splicing of LOXL2 to enhance the production of LOXL2Δ13.

Lysyl oxidase-like 2 (LOXL2) is a member of the lysyl oxidase family and has the ability to catalyze the cross-linking of extracellular matrix collagen and elastin. High expression of LOXL2 is related to tumor cell proliferation, invasion, and metastasis. LOXL2 contains 14 exons. Previous studies have found that LOXL2 has abnormal alternative splicing and exon skipping in a variety of tissues and cells, resulting in a new alternatively spliced isoform denoted LOXL2Δ13. LOXL2Δ13 lacks LOXL2WT exon 13, but its encoded protein has greater ability to induce tumor cell proliferation, invasion, and metastasis. However, the molecular events that produce LOXL2Δ13 are still unclear. In this study, we found that overexpression of the splicing factor hnRNPA1 in cells can regulate the alternative splicing of LOXL2 and increase the expression of LOXL2Δ13. The exonic splicing silencer exists at the 3' splice site and 5' splice site of LOXL2 exon 13. HnRNPA1 can bind to the exonic splicing silencer and inhibit the inclusion of exon 13. The RRM domain of hnRNPA1 and phosphorylation of hnRNPA1 at S91 and S95 are important for the regulation of LOXL2 alternative splicing. These results show that hnRNPA1 is a splicing factor that enhances the production of LOXL2Δ13.

Serine/threonine-protein kinase D2-mediated phosphorylation of DSG2 threonine 730 promotes esophageal squamous cell carcinoma progression.

Desmoglein-2 (DSG2) is a transmembrane glycoprotein belonging to the desmosomal cadherin family, which mediates cell-cell junctions; regulates cell proliferation, migration, and invasion; and promotes tumor development and metastasis. We previously showed serum DSG2 to be a potential biomarker for the diagnosis of esophageal squamous cell carcinoma (ESCC), although the significance and underlying molecular mechanisms were not identified. Here, we found that DSG2 was increased in ESCC tissues compared with adjacent tissues. In addition, we demonstrated that DSG2 promoted ESCC cell migration and invasion. Furthermore, using interactome analysis, we identified serine/threonine-protein kinase D2 (PRKD2) as a novel DSG2 kinase that mediates the phosphorylation of DSG2 at threonine 730 (T730). Functionally, DSG2 promoted ESCC cell migration and invasion dependent on DSG2-T730 phosphorylation. Mechanistically, DSG2 T730 phosphorylation activated EGFR, Src, AKT, and ERK signaling pathways. In addition, DSG2 and PRKD2 were positively correlated with each other, and the overall survival time of ESCC patients with high DSG2 and PRKD2 was shorter than that of patients with low DSG2 and PRKD2 levels. In summary, PRKD2 is a novel DSG2 kinase, and PRKD2-mediated DSG2 T730 phosphorylation promotes ESCC progression. These findings may facilitate the development of future therapeutic agents that target DSG2 and DSG2 phosphorylation. © 2024 The Pathological Society of Great Britain and Ireland.

KAT8/SIRT7-mediated Fascin-K41 acetylation/deacetylation regulates tumor metastasis in esophageal squamous cell carcinoma.

Fascin actin-bundling protein 1 (Fascin) is highly expressed in a variety of cancers, including esophageal squamous cell carcinoma (ESCC), working as an important oncogenic protein and promoting the migration and invasion of cancer cells by bundling F-actin to facilitate the formation of filopodia and invadopodia. However, it is not clear how exactly the function of Fascin is regulated by acetylation in cancer cells. Here, in ESCC cells, the histone acetyltransferase KAT8 catalyzed Fascin lysine 41 (K41) acetylation, to inhibit Fascin-mediated F-actin bundling and the formation of filopodia and invadopodia. Furthermore, NAD-dependent protein deacetylase sirtuin (SIRT) 7-mediated deacetylation of Fascin-K41 enhances the formation of filopodia and invadopodia, which promotes the migration and invasion of ESCC cells. Clinically, the analysis of cancer and adjacent tissue samples from patients with ESCC showed that Fascin-K41 acetylation was lower in the cancer tissue of patients with lymph node metastasis than in that of patients without lymph node metastasis, and low levels of Fascin-K41 acetylation were associated with a poorer prognosis in patients with ESCC. Importantly, K41 acetylation significantly blocked NP-G2-044, one of the Fascin inhibitors currently being clinically evaluated, suggesting that NP-G2-044 may be more suitable for patients with low levels of Fascin-K41 acetylation, but not suitable for patients with high levels of Fascin-K41 acetylation. © 2024 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Dephosphorylation of T517 on Hemocyanin Is Required for Antibacterial Activity in Penaeus vannamei.

Posttranslational modifications expand the functions of immune-related proteins, especially during infections. The respiratory glycoprotein, hemocyanin, has been implicated in many other functions, but the role of phosphorylation modification in its functional diversity is not fully understood. In this study, we show that Penaeus vannamei hemocyanin (PvHMC) undergoes phosphorylation modification during bacterial infection. Dephosphorylation of PvHMC mediated by P. vannamei protein phosphatase 2A catalytic increases its in vitro antibacterial activity, whereas phosphorylation by P. vannamei casein kinase 2 catalytic subunit α decreases its oxygen-carrying capacity and attenuates its in vitro antibacterial activity. Mechanistically, we show that Thr517 is a critical phosphorylation modification site on PvHMC to modulate its functions, which when mutated attenuates the action of P. vannamei casein kinase 2 catalytic subunit α and P. vannamei protein phosphatase 2A catalytic, and hence abolishes the antibacterial activity of PvHMC. Our results reveal that phosphorylation of PvHMC modulates its antimicrobial functions in penaeid shrimp.

Prenylated PALM2 Promotes the Migration of Esophageal Squamous Cell Carcinoma Cells Through Activating Ezrin.

Proteins containing a CAAX motif at the C-terminus undergo prenylation for localization and activity and include a series of key regulatory proteins, such as RAS superfamily members, heterotrimeric G proteins, nuclear lamina protein, and several protein kinases and phosphatases. However, studies of prenylated proteins in esophageal cancer are limited. Here, through research on large-scale proteomic data of esophageal cancer in our laboratory, we found that paralemmin-2 (PALM2), a potential prenylated protein, was upregulated and associated with poor prognosis in patients. Low-throughput verification showed that the expression of PALM2 in esophageal cancer tissues was higher than that in their paired normal esophageal epithelial tissues, and it was generally expressed in the membrane and cytoplasm of esophageal cancer cells. PALM2 interacted with the two subunits of farnesyl transferase (FTase), FNTA and FNTB. Either the addition of an FTase inhibitor or mutation in the CAAX motif of PALM2 (PALM2C408S) impaired its membranous localization and reduced the membrane location of PALM2, indicating PALM2 was prenylated by FTase. Overexpression of PALM2 enhanced the migration of esophageal squamous cell carcinoma cells, whereas PALM2C408S lost this ability. Mechanistically, PALM2 interacted with the N-terminal FERM domain of ezrin of the ezrin/radixin/moesin (ERM) family. Mutagenesis indicated that lysine residues K253/K254/K262/K263 in ezrin's FERM domain and C408 in PALM2's CAAX motif were important for PALM2/ezrin interaction and ezrin activation. Knockout of ezrin prevented enhanced cancer cell migration by PALM2 overexpression. PALM2, depending on its prenylation, increased both ezrin membrane localization and phosphorylation of ezrin at Y146. In summary, prenylated PALM2 enhances the migration of cancer cells by activating ezrin.

Sulconazole Induces PANoptosis by Triggering Oxidative Stress and Inhibiting Glycolysis to Increase Radiosensitivity in Esophageal Cancer.

Esophageal cancer is the seventh most common cancer in the world. Although traditional treatment methods such as radiotherapy and chemotherapy have good effects, their side effects and drug resistance remain problematic. The repositioning of drug function provides new ideas for the research and development of anticancer drugs. We previously showed that the Food and Drug Administration-approved drug sulconazole can effectively inhibit the growth of esophageal cancer cells, but its molecular mechanism is not clear. Here, our study demonstrated that sulconazole had a broad spectrum of anticancer effects. It can not only inhibit the proliferation but also inhibit the migration of esophageal cancer cells. Both transcriptomic sequencing and proteomic sequencing showed that sulconazole could promote various types of programmed cell death and inhibit glycolysis and its related pathways. Experimentally, we found that sulconazole induced apoptosis, pyroptosis, necroptosis, and ferroptosis. Mechanistically, sulconazole triggered mitochondrial oxidative stress and inhibited glycolysis. Finally, we showed that low-dose sulconazole can increase radiosensitivity of esophageal cancer cells. Taken together, these new findings provide strong laboratory evidence for the clinical application of sulconazole in esophageal cancer.

CRdb: a comprehensive resource for deciphering chromatin regulators in human.

Chromatin regulators (CRs) regulate epigenetic patterns on a partial or global scale, playing a critical role in affecting multi-target gene expression. As chromatin immunoprecipitation sequencing (ChIP-seq) data associated with CRs are rapidly accumulating, a comprehensive resource of CRs needs to be built urgently for collecting, integrating, and processing these data, which can provide abundant annotated information on CR upstream and downstream regulatory analyses as well as CR-related analysis functions. This study established an integrative CR resource, named CRdb (http://cr.liclab.net/crdb/), with the aim of curating a large number of available resources for CRs and providing extensive annotations and analyses of CRs to help biological researchers clarify the regulation mechanism and function of CRs. The CRdb database comprised a total of 647 CRs and 2,591 ChIP-seq samples from more than 300 human tissues and cell types. These samples have been manually curated from NCBI GEO/SRA and ENCODE. Importantly, CRdb provided the abundant and detailed genetic annotations in CR-binding regions based on ChIP-seq. Furthermore, CRdb supported various functional annotations and upstream regulatory information on CRs. In particular, it embedded four types of CR regulatory analyses: CR gene set enrichment, CR-binding genomic region annotation, CR-TF co-occupancy analysis, and CR regulatory axis analysis. CRdb is a useful and powerful resource that can help in exploring the potential functions of CRs and their regulatory mechanism in diseases and biological processes.

Application of Sodium-Rich Multifunctional Hard Carbon Synthesized via Multi-Alloy Grafting Strategy for Presodiation in High-Performance Sodium-Ion Batteries.

In sodium-ion pouch batteries based on hard carbon, an additional source of active sodium significantly enhances the battery's initial coulombic efficiency and compensates for the loss of active sodium ions during cycling. This study investigates the interaction between metallic sodium with carbon materials and develops a composite powder material of sodium-rich lithium-aluminum using a multi-alloy grafting strategy, to replenish the initial loss of active sodium in the hard carbon materials. To enhance the stability and utilization of this highly active sodium source, a novel slurry system based on polyethylene oxide (PEO) as a binder and dimethyl carbonate (DMC) as a solvent is introduced. Furthermore, this study designs a hard carbon composite electrode structure with a stable layer and sacrificial layer (NPH), which not only accommodates current battery processing environments but also demonstrates excellent potential in practical applications. Ultimately, the soft-packed sodium-ion battery consists of NPH electrodes with composite sodium ferric pyrophosphate (NFPP) and demonstrates excellent initial coulombic efficiency (91%) and ultra-high energy density (205 Wh kg-1). These results indicate significant technological and application implications for future energy storage.

Discovery of octahydropyrrolo [3,2-b] pyridin derivative as a highly selective Type I inhibitor of FGFR3 over VEGFR2 by high-throughput virtual screening.

Although the aberrant activity of fibroblast growth factor receptor 3 (FGFR3) is implicated in various cancers, the reported kinase inhibitors of FGFR3 tend to cause side effects resulting from the inhibitory activity on vascular endothelial growth factor receptor 2 (VEGFR2). Therefore, it is necessary to find a novel high-selective inhibitor of FGFR3 over VEGFR2 from the small-molecule compound database. In this study, integrated virtual screening protocols were established to screen for selective inhibitors of FGFR3 over VEGFR2 in Drugbank and Asinex databases by combining three-dimensional pharmacophore model, molecular docking, molecular dynamics (MD) simulation, and molecular mechanics Poisson-Boltzmann surface area (MMPBSA) calculations. Finally, it is found that Asinex-5082, as an octahydropyrrolo[3,2-b] pyridin derivative, has larger binding free energy with FGFR3 (-39.3 kcal/mol) than reference drug Erdafitinib (-29.9 kcal/mol), while cannot bind with VEGFR2, resulting in considerable inhibitory selectivity. This is because Asinex-5082, unlike Erdafitinib, has not m-dimethoxybenzene with large steric hindrance, thus can enter the larger ATP-binding pocket of FGFR3 with DFG-in conformation to form hydrophobic interaction with residues Met529, Ile539, and Tyr557 as well as hydrogen bond with Ala558. On the other hand, due to the fact that the benzodioxane and N-heterocyclic rings are connected by carbonyl (C=O), Asinex-5082 cannot rotate freely so as to enter the smaller ATP binding pocket of VEGFR2 on the DFG-out conformation. The lead molecule Asinex-5082 may facilitate the rational design and development of novel selective inhibitors of FGFR3 over VEGFR2 as anticancer drugs.

Molecular dynamics simulation study on the structures of fascin mutants.

Fascin is a filamentous actin (F-actin) bundling protein, which cross-links F-actin into bundles and becomes an important component of filopodia on the cell surface. Fascin is overexpressed in many types of cancers. The mutation of fascin affects its ability to bind to F-actin and the progress of cancer. In this paper, we have studied the effects of residues of K22, K41, K43, K241, K358, K399, and K471 using molecular dynamics (MD) simulation. For the strong-effect residues, that is, K22, K41, K43, K358, and K471, our results show that the mutation of K to A leads to large values of root mean square fluctuation (RMSF) around the mutated residues, indicating those residues are important for the flexibility and thermal stability. On the other hand, based on residue cross-correlation analysis, alanine mutations of these residues reinforce the correlation between residues. Together with the RMSF data, the local flexibility is extended to the entire protein by the strong correlations to influence the dynamics and function of fascin. By contrast, for the mutants of K241A and K399A those do not affect the function of fascin, the RMSF data do not show significant differences compared with wild-type fascin. These findings are in a good agreement with experimental studies.

TRlnc: a comprehensive database for human transcriptional regulatory information of lncRNAs.

Long noncoding RNAs (lncRNAs) have been proven to play important roles in transcriptional processes and biological functions. With the increasing study of human diseases and biological processes, information in human H3K27ac ChIP-seq, ATAC-seq and DNase-seq datasets is accumulating rapidly, resulting in an urgent need to collect and process data to identify transcriptional regulatory regions of lncRNAs. We therefore developed a comprehensive database for human regulatory information of lncRNAs (TRlnc, http://bio.licpathway.net/TRlnc), which aimed to collect available resources of transcriptional regulatory regions of lncRNAs and to annotate and illustrate their potential roles in the regulation of lncRNAs in a cell type-specific manner. The current version of TRlnc contains 8 683 028 typical enhancers/super-enhancers and 32 348 244 chromatin accessibility regions associated with 91 906 human lncRNAs. These regions are identified from over 900 human H3K27ac ChIP-seq, ATAC-seq and DNase-seq samples. Furthermore, TRlnc provides the detailed genetic and epigenetic annotation information within transcriptional regulatory regions (promoter, enhancer/super-enhancer and chromatin accessibility regions) of lncRNAs, including common SNPs, risk SNPs, eQTLs, linkage disequilibrium SNPs, transcription factors, methylation sites, histone modifications and 3D chromatin interactions. It is anticipated that the use of TRlnc will help users to gain in-depth and useful insights into the transcriptional regulatory mechanisms of lncRNAs.

ASO Author Reflections: A Multiomic Novel Staging System for Esophageal Squamous Cell Carcinoma Patients.

The past eighth edition of the American Joint Committee on Cancer (AJCC)/International Union against Cancer (UICC) pathologic tumor-node-metastasis (pTNM) staging system for esophageal squamous cell carcinoma (ESCC) patients, which also is the gold standard of postoperative treatment decision-making, needs to be continuously improved. To improve the efficiency of the staging system, the proteomic data from Chinese ESCC patients was combined with preoperative radiomic data and pTNM data to establish the multiomic RadpTNM and ProtRadpTNM models and compare them with the traditional pTNM staging system. The results suggest that both the RadpTNM and ProtRadpTNM models are significantly better than the traditional pTNM staging system. Future prospective multicentered cohort studies in Asian and Caucasian patients with ESCC are warranted to evaluate the efficiency of the multiomic models.

Development and Validation of a New Staging System for Esophageal Squamous Cell Carcinoma Patients Based on Combined Pathological TNM, Radiomics, and Proteomics.

OBJECTIVE: This study aimed to construct a new staging system for patients with esophageal squamous cell carcinoma (ESCC) based on combined pathological TNM (pTNM) stage, radiomics, and proteomics. METHODS: This study collected patients with radiomics and pTNM stage (Cohort 1, n = 786), among whom 103 patients also had proteomic data (Cohort 2, n = 103). The Cox regression model with the least absolute shrinkage and selection operator, and the Cox proportional hazards model were used to construct a nomogram and predictive models. Concordance index (C-index) and the integrated area under the time-dependent receiver operating characteristic (ROC) curve (IAUC) were used to evaluate the predictive models. The corresponding staging systems were further assessed using Kaplan-Meier survival curves. RESULTS: For Cohort 1, the RadpTNM4c staging systems, constructed based on combined pTNM stage and radiomic features, outperformed the pTNM4c stage in both the training dataset 1 (Train1; IAUC 0.711 vs. 0.706, p < 0.001) and the validation dataset 1 (Valid1; IAUC 0.695 vs. 0.659, p < 0.001; C-index 0.703 vs. 0.674, p = 0.029). For Cohort 2, the ProtRadpTNM2c staging system, constructed based on combined pTNM stage, radiomics, and proteomics, outperformed the pTNM2c stage in both the Train2 (IAUC 0.777 vs. 0.610, p < 0.001; C-index 0.898 vs. 0.608, p < 0.001) and Valid2 (IAUC 0.746 vs. 0.608, p < 0.001; C-index 0.889 vs. 0.641, p = 0.009) datasets. CONCLUSIONS: The ProtRadpTNM2c staging system, based on combined pTNM stage, radiomic, and proteomic features, improves the predictive performance of the classical pTNM staging system.

Resonance-Enhanced Emission Effects toward Dual-State Emissive Bright Red and Near-Infrared Emitters.

Resonance-enhanced emission (REE) effect was discovered and lead to a novel dye family of hydrostyryl pyridinium derivatives in our recent work. Herein, the REE effect was employed to design a red and near-infrared dual-state emissive fluorophore family of SW-OH-NO2 derivatives which were easily synthesized by coupling an electron-withdrawing group (W) onto nitro(hydroxyl)styryl (S-OH-NO2 ) through a C=C double bond as π-bridge. The deprotonation of a phenolic hydroxyl group promoted by a nitro group and the electron-withdrawing group (W) on the other side of the π-bridge triggered resonance, resulting in significantly red-shifted emission. All the resultant SW-OH-NO2 compounds showed excellent dual-state emission behavior. Remarkably, hydrostyryl quinolinium (SQ-OH-NO2 ) is one of the smallest NIR emitter molecular skeleton (λem =725 nm, MW<400) and showed dual-state emission characteristics and obvious viscosity-depended fluorescent behaviors. In addition to constructing electron donor-acceptor structures and prolonging π-bridges, the REE effect promises a reliable strategy toward novel fluorophores with small size, long emissive wavelength, and dual-emission characteristics, and importantly, feasible industrial manufactures and applications due to their easy and low-cost synthesis strategy.

The possibilities of LOXL4 as a prognostic marker for carcinomas.

Lysyl oxidase-like 4 (LOXL4), a member of lysyl oxidase family, is a copper and lysine tyrosylquinone-dependent amine oxidase that serves the role of catalyzing the cross-linking of elastin and collagen in the extracellular matrix. Numerous studies have shown a significant association between LOXL4 expression levels and tumor proliferation, migration, invasion and patients' prognosis and overall survival in different types of tumors. Here we review their relationship and the molecular pathogenesis behind them, aiming to explore the possibilities of LOXL4 as a prognostic marker for diverse carcinomas and provide some indications for further research in this field.

Systematic Proteome and Lysine Succinylome Analysis Reveals Enhanced Cell Migration by Hyposuccinylation in Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) is an aggressive malignancy with poor therapeutic outcomes. However, the alterations in proteins and posttranslational modifications (PTMs) leading to the pathogenesis of ESCC remain unclear. Here, we provide the comprehensive characterization of the proteome, phosphorylome, lysine acetylome, and succinylome for ESCC and matched control cells using quantitative proteomic approach. We identify abnormal protein and PTM pathways, including significantly downregulated lysine succinylation sites in cancer cells. Focusing on hyposuccinylation, we reveal that this altered PTM was enriched on enzymes of metabolic pathways inextricably linked with cancer metabolism. Importantly, ESCC malignant behaviors such as cell migration are inhibited once the level of succinylation was restored in vitro or in vivo. This effect was further verified by mutations to disrupt succinylation sites in candidate proteins. Meanwhile, we found that succinylation has a negative regulatory effect on histone methylation to promote cancer migration. Finally, hyposuccinylation is confirmed in primary ESCC specimens. Our findings together demonstrate that lysine succinylation may alter ESCC metabolism and migration, providing new insights into the functional significance of PTM in cancer biology.

Activation of bivalent factor DLX5 cooperates with master regulator TP63 to promote squamous cell carcinoma.

To reconstruct systematically hyperactive transcription factor (TF)-dependent transcription networks in squamous cell carcinomas (SCCs), a computational method (ELMER) was applied to 1293 pan-SCC patient samples, and 44 hyperactive SCC TFs were identified. As a top candidate, DLX5 exhibits a notable bifurcate re-configuration of its bivalent promoter in cancer. Specifically, DLX5 maintains a bivalent state in normal tissues; its promoter is hypermethylation, leading to DLX5 transcriptional silencing in esophageal adenocarcinoma (EAC). In stark contrast, DLX5 promoter gains active histone marks and becomes transcriptionally activated in ESCC, which is directly mediated by SOX2. Functionally, silencing of DLX5 substantially inhibits SCC viability both in vitro and in vivo. Mechanistically, DLX5 cooperates with TP63 in regulating ∼2000 enhancers and promoters, which converge on activating cancer-promoting pathways. Together, our data establish a novel and strong SCC-promoting factor and elucidate a new epigenomic mechanism - bifurcate chromatin re-configuration - during cancer development.

The Expression Pattern and Clinical Significance of Lysyl Oxidase Family in Gliomas.

LOX (Lysyl oxidase) family participates in the catalysis of collagen and elastin to maintain ECM homeostasis. Glioma is the most common primary brain tumor and LOX family has not been systemic studied in glioma. In this study, we found LOX family members are upregulated expressed in gliomas samples. A protein-protein interaction network (PPIN) was construct to visualize and understand the differential expression pattern, as well as functional annotation, for LOX family and their interacting proteins, which involved in collagen fibril organization and MAPK signaling pathway. Through subcellular localization distribution, the LOX family members distribute both intracellular and extracellular. All five LOX members are consistently significantly correlate with dendritic cell both in immune infiltrate of GBM and LGG. Survival analysis showed that high expression of LOX family is associated with a poor prognosis of gliomas patients. These analyses provide important clues to identify the potential biological roles for LOX family in gliomas, which might serve as diagnosis markers.

HiFreSP: A novel high-frequency sub-pathway mining approach to identify robust prognostic gene signatures.

With the increasing awareness of heterogeneity in cancers, better prediction of cancer prognosis is much needed for more personalized treatment. Recently, extensive efforts have been made to explore the variations in gene expression for better prognosis. However, the prognostic gene signatures predicted by most existing methods have little robustness among different datasets of the same cancer. To improve the robustness of the gene signatures, we propose a novel high-frequency sub-pathways mining approach (HiFreSP), integrating a randomization strategy with gene interaction pathways. We identified a six-gene signature (CCND1, CSF3R, E2F2, JUP, RARA and TCF7) in esophageal squamous cell carcinoma (ESCC) by HiFreSP. This signature displayed a strong ability to predict the clinical outcome of ESCC patients in two independent datasets (log-rank test, P = 0.0045 and 0.0087). To further show the predictive performance of HiFreSP, we applied it to two other cancers: pancreatic adenocarcinoma and breast cancer. The identified signatures show high predictive power in all testing datasets of the two cancers. Furthermore, compared with the two popular prognosis signature predicting methods, the least absolute shrinkage and selection operator penalized Cox proportional hazards model and the random survival forest, HiFreSP showed better predictive accuracy and generalization across all testing datasets of the above three cancers. Lastly, we applied HiFreSP to 8137 patients involving 20 cancer types in the TCGA database and found high-frequency prognosis-associated pathways in many cancers. Taken together, HiFreSP shows higher prognostic capability and greater robustness, and the identified signatures provide clinical guidance for cancer prognosis. HiFreSP is freely available via GitHub: https://github.com/chunquanlipathway/HiFreSP.