Oncogenesis mimics key aspects of embryonic development. However, the underlying mechanisms are incompletely understood. Here, we demonstrate that the splicing events specifically active during human organogenesis, are broadly reactivated in the organ-specific tumor. Such events are associated with key oncogenic processes and predict proliferation rates in cancer cell lines as well as patient survival. Such events preferentially target nitrosylation and transmembrane-region domains, whose coordinated splicing in multiple genes respectively affect intracellular transport and N-linked glycosylation. We infer critical splicing factors potentially regulating embryonic splicing events and show that such factors are potential oncogenic drivers and are upregulated specifically in malignant cells. Multiple complementary analyses point to MYC and FOXM1 as potential transcriptional regulators of critical splicing factors in brain and liver. Our study provides a comprehensive demonstration of a splicing-mediated link between development and cancer, and suggest anti-cancer targets including splicing events, and their upstream splicing and transcriptional regulators.

The small GTPase family is well-studied in cancer and cellular physiology. With 162 annotated human genes, the family has a broad expression throughout cells of the body. Members of the family have multiple exons that require splicing. Yet, the role of splicing within the family has been underexplored. We have studied the splicing dynamics of small GTPases throughout 41,671 samples by integrating Nanopore and Illumina sequencing techniques. Within this work, we have made several discoveries. 1). Using the GTEx long read data of 92 samples, each small GTPase gene averages two transcripts, with 83 genes (51%) expressing two or more isoforms. 2). Cross-tissue analysis of GTEx from 17,382 samples shows 41 genes (25%) expressing two or more protein-coding isoforms. These include protein-changing transcripts in genes such as RHOA, RAB37, RAB40C, RAB4B, RAB5C, RHOC, RAB1A, RAN, RHEB, RAC1, and KRAS. 3). The isolation and library technique of the RNAseq influences the abundance of non-sense-mediated decay and retained intron transcripts of small GTPases, which are observed more often in genes than appreciated. 4). Analysis of 16,243 samples of "Blood PAXgene" identified seven genes (3.7%; RHOA, RAB40C, RAB4B, RAB37, RAB5B, RAB5C, RHOC) with two or more transcripts expressed as the major isoform (75% of the total gene), suggesting a role of genetics in altering splicing. 5). Rare (ARL6, RAB23, ARL13B, HRAS, NRAS) and common variants (GEM, RHOC, MRAS, RAB5B, RERG, ARL16) can influence splicing and have an impact on phenotypes and diseases. 6). Multiple genes (RAB9A, RAP2C, ARL4A, RAB3A, RAB26, RAB3C, RASL10A, RAB40B, and HRAS) have sex differences in transcript expression. 7). Several exons are included or excluded for small GTPase genes (RASEF, KRAS, RAC1, RHEB, ARL4A, RHOA, RAB30, RHOBTB1, ARL16, RAP1A) in one or more forms of cancer. 8). Ten transcripts are altered in hypoxia (SAR1B, IFT27, ARL14, RAB11A, RAB10, RAB38, RAN, RIT1, RAB9A) with RHOA identified to have a transient 3'UTR RNA base editing at a conserved site found in all of its transcripts. Overall, we show a remarkable and dynamic role of splicing within the small GTPase family that requires future explorations.

Gastric cancer heterogeneity represents a barrier to disease management. We generated a comprehensive single-cell atlas of gastric cancer (>200,000 cells) comprising 48 samples from 31 patients across clinical stages and histologic subtypes. We identified 34 distinct cell-lineage states including novel rare cell populations. Many lineage states exhibited distinct cancer-associated expression profiles, individually contributing to a combined tumor-wide molecular collage. We observed increased plasma cell proportions in diffuse-type tumors associated with epithelial-resident KLF2 and stage-wise accrual of cancer-associated fibroblast subpopulations marked by high INHBA and FAP coexpression. Single-cell comparisons between patient-derived organoids (PDO) and primary tumors highlighted inter- and intralineage similarities and differences, demarcating molecular boundaries of PDOs as experimental models. We complemented these findings by spatial transcriptomics, orthogonal validation in independent bulk RNA-sequencing cohorts, and functional demonstration using in vitro and in vivo models. Our results provide a high-resolution molecular resource of intra- and interpatient lineage states across distinct gastric cancer subtypes.

We profiled gastric malignancies at single-cell resolution and identified increased plasma cell proportions as a novel feature of diffuse-type tumors. We also uncovered distinct cancer-associated fibroblast subtypes with INHBA-FAP-high cell populations as predictors of poor clinical prognosis. Our findings highlight potential origins of deregulated cell states in the gastric tumor ecosystem. This article is highlighted in the In This Issue feature, p. 587.

BACKGROUND Increasing evidence suggests that the alternative splicing (AS) signature plays a role in the carcinogenesis and prognosis of various cancers. However, the prognostic role of AS in gastric cancer is not clear and needs to be clarified. MATERIAL AND METHODS To identify the differentially expressed AS (DEAS) events, we performed a differential expression analysis between normal and tumor tissue. The DEAS event was further applied to construct a prognostic signature by performing univariate Cox regression analysis and least absolute shrinkage and selection operator (LASSO) analysis. The Kaplan-Meier curve analysis and receiver operating characteristic curve (ROC) analysis were used to evaluate the prognostic value of the AS signature. In addition, the network of the splicing events with splicing factors was constructed using the Cytoscape software. RESULTS A total of 30 005 alternative splicing (AS) events with 372 patients were retrieved from the SpliceSeq database and TCGA database. By performing differential expression analysis, a total of 419 alternative splicing events were screened out, including 56 upregulated and 363 downregulated. We further constructed an AS-related prognostic signature by conducting a series bioinformatics analyses. Moreover, we identified that the AS signature could serve as an independent predictor for the prognosis of GC. We also found that AS signature had a more robust and precise efficacy for prognostic prediction in GC patients. Interestingly, the areas under 3- and 5-year survival curves are similar, both of which are greater than 1-year survival curve, suggesting that the long-term predictive accuracy of our prognostic model built upon AS signature is superior. CONCLUSIONS We performed a comprehensive analysis of overall prognostic-associated AS events concerning GC and constructed a prognostic model to predict the long-term prognostic survival outcomes in GC patients. We also developed a network of splicing events with splicing factors to reveal new potential molecular diagnostic biomarkers and therapeutic targets for GC patients.

Gene expression and alternative splicing (AS) interact in complex ways to regulate biological process which is associated with cancer development. Here, by integrated analysis of gene expression and AS events, we aimed to identify the hub AS events and splicing factors relevant in gastric cancer development (GC). RNA-seq data, clinical data and AS events of 348 GC samples were obtained from the TCGA and TCGASpliceSeq databases. Cox univariable and multivariable analyses, KEGG and GO pathway analyses were performed to identify hub AS events and splicing factor/spliceosome genes, which were further validated in 53 GCs. By bioinformatics methods, we found that gene AS event- and gene expression-mediated GC progression shared the same mechanisms, such as PI3K/AKT pathway, but the involved genes were different. Though expression of 17 hub AS events were confirmed in 53 GC tissues, only 10 AS events in seven genes were identified as critical candidates related to GC progression, notably the AS events (Exon Skip) in CLSTN1 and SEC16A. Expression of these AS events in GC correlated with activation of the PI3K/AKT pathway. Genes with AS events associated with clinical parameters and prognosis were different from the genes whose mRNA levels were related to clinical parameters and prognosis. Besides, we further revealed that QKI and NOVA1 were the crucial splicing factors regulating expression of AS events in GC, but not spliceosome genes. Our integrated analysis revealed hub AS events in GC development, which might be the potential therapeutic targets for GC.

The well-known signal mediator and small GTPase family member, RHOA, has now been associated with the progression of specific malignancies. In this review, we appraise the biomedical literature regarding the role of this enzyme in gastric cancer (GC) signaling, suggesting potential clinical significance. To that end, we examined RHOA activity, with regard to second-generation hallmarks of cancer, finding particular association with the hallmark "activation of invasion and metastasis." Moreover, an abundance of studies show RHOA association with Lauren classification diffuse subtype, in addition to poorly differentiated GC. With regard to therapeutic value, we found RHOA signaling to influence the activity of specific widely used chemotherapeutics, and its possible antagonism by various dietary constituents. We also review currently available targeted therapies for GC. The latter, however, showed a paucity of such agents, underscoring the urgent need for further investigation into treatments for this highly lethal malignancy.

Voltage-gated sodium channels are involved in tumor metastasis, as potentiating or attenuating their activities affects the migration and invasion process of tumor cells. In the present study, we tested the effect of two peptide toxins, JZTX-I and HNTX-III which function as Nav1.7 activator and inhibitor, respectively, on the migration and invasion ability of prostate cancer (PCa) cell line Mat-LyLu. These two peptides showed opposite effects, and subsequently a comparative proteomic analysis characterized 64 differentially expressed membrane proteins from the JZTX-I- and HNTX-III-treated groups. Among these, 15 proteins were down-regulated and 49 proteins were up-regulated in the HNTX-III group. Bioinformatic analysis showed eight proteins are cytoskeleton proteins or related regulators, which might play important roles in the metastasis of Mat-LyLu cells. The altered expressions of four of these proteins, fascin, muskelin, annexin A2, and cofilin-1, were validated by western blot analysis. Further function network analysis of these proteins revealed that the Rho family GTPases RhoA and Rac1 might be of particular importance for the rat PCa cell invasion. Pharmacological data revealed that JZTX-I and HNTX-III could modulate the Rho signaling pathway in a Nav1.7-dependent manner. In summary, this study suggests that the Nav1.7-dependent regulation of Rho GTPase activity plays a vital role in Mat-LyLu cell migration and invasion and provides new insights into the treatment of PCa.

Gastric cancer is the third leading cause of cancer-related deaths and ranks as the fifth most common cancer worldwide. Incidence and mortality differ depending on the geographical region and gastric cancer ranks first in East Asian countries. Although genetic factors, gastric environment, and Helicobacter pylori infection have been associated with the pathogenicity and development of intestinal-type gastric cancer that follows the Correa's cascade, the pathogenicity of diffuse-type gastric cancer remains mostly unknown and undefined. However, genetic abnormalities in the cell adherence factors, such as E-cadherin and cellular activities that cause impaired cell integrity and physiology, have been documented as contributing factors. In recent years, H. pylori infection has been also associated with the development of diffuse-type gastric cancer. Therefore, in this report, we discuss the host factors as well as the bacterial factors that have been reported as associated factors contributing to the development of diffuse-type gastric cancer.