Nociceptive neurons interact directly with gastric cancer cells via a CGRP/Ramp1 axis to promote tumor progression.

Cancer cells have been shown to exploit neurons to modulate their survival and growth, including through establishment of neural circuits within the central nervous system (CNS) 1-3 . Here, we report a distinct pattern of cancer-nerve interactions between the peripheral nervous system (PNS) and gastric cancer (GC). In multiple GC mouse models, nociceptive nerves demonstrated the greatest degree of nerve expansion in an NGF-dependent manner. Neural tracing identified CGRP+ peptidergic neurons as the primary gastric sensory neurons. Three-dimensional co-culture models showed that sensory neurons directly connect with gastric cancer spheroids through synapse-like structures. Chemogenetic activation of sensory neurons induced the release of calcium into the cytoplasm of cancer cells, promoting tumor growth and metastasis. Pharmacological ablation of sensory neurons or treatment with CGRP inhibitors suppressed tumor growth and extended survival. Depolarization of gastric tumor membranes through in vivo optogenetic activation led to enhanced calcium flux in nodose ganglia and CGRP release, defining a cancer cell-peptidergic neuronal circuit. Together, these findings establish the functional connectivity between cancer and sensory neurons, identifying this pathway as a potential therapeutic target.

An optimized protocol for isolation of murine pancreatic single cells with high yield and purity.

Here, we present a protocol for rapidly isolating single cells from the mouse pancreas, minimizing damage caused by digestive enzymes in exocrine cells. We guide you through steps to optimize the dissection sequence, enzyme composition, and operational procedures, resulting in high yields of viable pancreatic single cells. This protocol can be applied across a wide range of research areas, including single-cell sequencing, gene expression profiling, primary cell culture, and even the development of spheroids or organoids. For complete details on the use and execution of this protocol, please refer to Jiang et al. (2023).1.

Peptidome analysis reveals critical roles for peptides in a rat model of intestinal ischemia/reperfusion injury.

Intestinal ischemia/reperfusion injury (IIRI) has the potential to be life threatening and is associated with significant morbidity and serious damage to distant sites in the body on account of disruption of the intestinal mucosal barrier. In the present study, we have explored this line of research by comparing and identifying peptides that originated from the intestinal segments of IIRI model rats by using liquid chromatography-mass spectrometry (LC-MS). We also analyzed the basic characteristics, cleavage patterns, and functional domains of differentially expressed peptides (DEPs) between the IIRI model rats and control (sham-operated) rats and identified bioactive peptides that are potentially associated with ischemia reperfusion injury. We also performed bioinformatics analyses in order to identify the biological roles of the DEPs based on their precursor proteins. Enrichment analysis demonstrated the role of several DEPs in impairment of the intestinal mucosal barrier caused by IIRI. Based on the results of comprehensive ingenuity pathway analysis, we identified the DEPs that were significantly correlated with IIRI. We identified a candidate precursor protein (Actg2) and seven of its peptides, and we found that Actg2-6 had a more significant difference in its expression, a longer half-life, and better lipophilicity, hydrophobicity, and stability than the other candidate Actg2 peptides examined. Furthermore, we observed that Actg2-6 might play critical roles in the protection of the intestinal mucosal barrier during IIRI. In summary, our study provides a better understanding of the peptidomics profile of IIRI, and the results indicate that Actg2-6 could be a useful target in the treatment of IIRI.

Ubiquitin-specific peptidase 24 accelerates aerobic glycolysis and tumor progression in gastric carcinoma through stabilizing PLK1 to activate NOTCH1.

Ubiquitin-specific peptidase 24 (USP24), a member of the deubiquitinase family, plays an important role in tumor regulation. However, the role of USP24 in gastric cancer (GC) is unknown. The aim of our study was to explore the role of USP24 in GC to seek new therapeutic targets for GC. TCGA analysis showed that USP24 was upregulated in GC patients, and its high expression levels were associated with poor prognosis. It was found that overexpressed USP24 promoted GC cell proliferation and abnormal glycolysis. Further analysis and study showed that USP24 could promote the stability and increase the expression of oncogene PLK1. Knocking down USP24 can reduce the stability of PLK1 to reduce Notch 1 activity, thus inhibiting GC glycolysis, proliferation, and other processes and alleviating tumor progression. Knocking down USP24 can inhibit GC progression. In conclusion, USP24 was upregulated in GC and promoted the growth and glycolysis of GC cells, the mechanism of which was related to the deubiquitination of PLK1 and the increase of its stability. Silencing USP24 inhibited the GC process. This study suggests that the USP24/PLK1/Notch 1 axis may be a novel therapeutic target for gastric cancer.

ADRB2 Regulates the Proliferation and Metastasis of Gastrointestinal Stromal Tumor Cells by Enhancing the ETV1-c-KIT Signaling.

Background: Gastrointestinal stromal tumor (GIST) originates from a pacemaker cell, the Cajal cell. However, little is known about the cancer neuroscience in GIST. In this study, we aimed to elucidate the clinical and biological roles of adrenoceptor beta 2 (ADRB2) in GIST. Methods: Immunohistochemistry was used to evaluate the expression of ADRB2 in GIST tissues. The biological effects of ADRB2 on GIST cell proliferation, migration, invasion, and apoptosis were explored using Cell Counting Kit -8, plate colony formation assay, transwell invasion assay, and flow cytometry. We also explored the growth and metastasis of xenograft tumors in nude mice. Western blotting was used to quantify protein expression and phosphorylation. Results: ADRB2 is generally highly expressed in GIST. High ADRB2 expression was significantly associated with risk level, tumor size, mitotic count, and metastasis. Overexpression of ADRB2 promoted GIST cell proliferation, migration, invasion, and apoptosis, while silencing ADRB2 expression showed the opposite effects. Furthermore, we found that silencing endogenous ADRB2 inhibited GIST progression and metastasis in nude mice. ADRB2-induced ETV1 upregulation enhanced the activation of c-KIT. Conclusion: ADRB2 plays an important role in the proliferation and metastasis of GIST and is expected to be a potential target for the treatment of GIST.

Retraction Notice to: miR-3174 Contributes to Apoptosis and Autophagic Cell Death Defects in Gastric Cancer Cells by Targeting ARHGAP10.

[This retracts the article DOI: 10.1016/j.omtn.2017.10.008.].

A Novel Pyroptosis-Associated Long Noncoding RNA Signature to Predict the Prognosis of Patients with Colorectal Cancer.

PURPOSE: Pyroptosis plays an important role in tumor progression. However, there is no pyroptosis-associated long noncoding RNA (lncRNA) signature to predict the prognosis of patients with colorectal cancer (CRC). MATERIALS AND METHODS: The RNA sequencing data (RNA-seq) and corresponding clinical information relating to CRC patients were obtained from the Cancer Genome Atlas (TCGA) database and the GSE39582 dataset. Univariate Cox regression analysis was used to identify pyroptosis-associated lncRNAs linked to CRC prognosis. Subsequently, multivariate Cox regression analysis was performed to construct a pyroptosis-associated lncRNAs signature within the TCGA cohort, which was then validated using the GSE39582 dataset. We used Kaplan-Meier (K-M) analysis, principal component analysis (PCA), and receiver operating characteristic curve (ROC) analysis to evaluate our novel lncRNA signature. Finally, gene set enrichment analysis (GSEA) was performed to explore the potential function of the lncRNA signature. RESULTS: We constructed a pyroptosis-associated lncRNA signature comprising four lncRNAs (ELFN1-AS1, PCAT6, TNRC6C-AS1, and ZEB1-AS1). CRC patients were subdivided into high- and low-risk groups based on median risk scores. The results of the K-M, PCA, and ROC analyses showed that this signature could accurately predict the prognosis of CRC patients. Univariate and multivariate Cox regression analyses showed that the pyroptosis-associated signature was an independent prognostic factor. Functional analysis suggested that tumor-associated pathways were enriched for in the high-risk CRC patient group. CONCLUSION: Our study established an effective prognostic signature for CRC patients that may represent a potential therapeutic target.

Involvement of APRIL in Helicobacter pylori-related gastric cancer.

BACKGROUND/AIMS: A proliferation-inducing ligand (APRIL, also known as TNFSF13, CD256) is a member of the tumor necrosis factor (TNF) superfamily and involved in a diverse set of diseases. In this work, we explored the potential associations and underlying mechanism in patients suffered from gastric cancer between the expression of APRIL and H. pylori infection. METHODS: We analyzed APRIL expression levels in 200 GC tissue samples by immunohistochemistry staining. H. pylori infection was detected by modified Giemsa staining. The biological effects of APRIL on human GC cells in vitro and in vivo were tested by CCK-8 assay, colony formation, flow cytometry detection, transwell migration assay, matrigel invasion assay, and tumor xenograft assay in animals. RESULTS: APRIL reactivity was positively correlated with H. pylori infection in vitro and vivo. It turned out that the decrease of miR-145 expression was dose-dependent and time-dependent on H. pylori infection and in consistent with APRIL expression. MiR-145 significantly attenuated the effect of H. pylori infection on APRIL gene expression in SGC7901 and BGC823 cell lines. Furthermore, APRIL overexpression promoted the proliferation, migration, invasion, and transfer of GC cells and decreased apoptosis, while APRIL knockdown suppressed these effects. We confirmed that APRIL activated the canonical NF-κB pathway through phosphorylation of AKT. CONCLUSION: The expression of APRIL, which promoted the proliferation, migration, invasion, viability, and metastasis of GC cells, was upregulated in human H. pylori-infected GC through miR-145. Besides, APRIL-induced gastric tumorigenicity via activating NF-κB pathway. These results may provide a framework for the deeper analysis of APRIL in GC risk and prognosis.

KIF15 promotes the evolution of gastric cancer cells through inhibition of reactive oxygen species-mediated apoptosis.

Kinesin family member 15 (KIF15) is a member of the kinesin superfamily of proteins, which promotes cell mitosis, participates in the transport of intracellular materials, and helps structural assembly and cell signaling pathways transduction. However, its biological role and molecular mechanisms of action in the development of gastric cancer (GC) remain unclear. In the present study, an integrated analysis of The Cancer Genome Atlas (TCGA), Gene Expression Omnibus database, and Kaplan-Meier plotter database was performed to predict the expression and prognostic value of KIF15 in GC patients. Detection of KIF15 expression in GC cells and tissues was performed by a quantitative polymerase chain reaction. In vitro cell proliferation, viability, colony formation ability and flow cytometry assays, and in vivo tumorigenicity assay, were performed to evaluate the effects of KIF15 knockdown on GC cell phenotype. It was demonstrated that the expression of KIF15 messenger RNA in GC tissues was significantly higher compared with that in adjacent tissues, and was closely associated with larger tumor size and poor patient prognosis. In addition, functional studies demonstrated that, due to the increase in reactive oxygen species (ROS) generation, the interference with the expression of KIF15 not only decreased cell proliferation but also increased cell apoptosis and induced cell cycle arrest. ROS-mediated activation of c-Jun N-terminal kinase/c-Jun signaling reduced cell proliferation by regulating the GC cell cycle and increasing apoptosis. Taken together, the results of the present study indicate that KIF15 is an oncoprotein contributing to GC progression, and is expected to help identify novel biomarkers and treatment targets in GC.

Syndecan-2 in colorectal cancer plays oncogenic role via epithelial-mesenchymal transition and MAPK pathway.

PURPOSE: In this study, we aimed to elucidate the biological roles of Syndecan-2 (SDC2) in colorectal cancer (CRC), thereby further understanding its clinical role. METHODS: The expression of SDC2 was assessed by qRT-PCR and Western blot analysis. To understand the potential biological role of SDC2, we also explored the correlation between its expression level and clinicopathologic parameters. By using MTT, plate colony formation assay, Transwell invasion assays, and flow cytometry in vitro, the biological impact of SDC2 on CRC cell proliferation, migration, invasion, and apoptosis. In addition, the related signaling pathways were investigated. RESULTS: SDC2 expression was significantly upregulated in CRC tissues. The expression of SDC2 was highly associated with four parameters, i.e., stage (P < 0.01), vascular invasion (P = 0.0045), lymph node metastasis (P=0.0018), and distant metastasis (P = 0.0019). Knockdown of SDC2 significantly reduced proliferation, migration, and invasion of HCT116 and SW480 cells, and induced cell apoptosis. Moreover, SDC2 promoted epithelial-mesenchymal transition (EMT) in CRC cells, whereas the ratio of p-MEK/MEK and p-ERK/ERK markedly reduced after depleting SDC2. CONCLUSION: During CRC development, overexpression of SDC2 plays a carcinogenic role in CRC. Therapeutic solutions targeting SDC2 may provide potential insights into CRC prevention and treatment.

Adrenergic modulation of AMPK­dependent autophagy by chronic stress enhances cell proliferation and survival in gastric cancer.

Epidemiological data show that chronic stress has adverse effects on the incidence and progression of cancer. As a critical target organ for stress hormones, the stomach is frequently subjected to stress­related injury. However, few reports regarding the association between stress and gastric cancer (GC) have been published. The present study aimed to investigate the effect of chronic stress on the growth and survival of GC, and the role of the autophagy process. A restraint­stress procedure over 21 days was used to establish a chronic stress mouse model. Subcutaneous xenografts and gastric orthotopic xenografts were established in BALB/c nude mice. Alzet osmotic minipumps containing either PBS or propranolol hydrochloride was inserted on the nape of the neck 7 days prior to the initiation of restraint stress. The presence of autophagosomes and autolysosomes were examined by electron microscopy. The stress hormone norepinephrine significantly enhanced the proliferation of GC cells. By inhibiting adrenoreceptor expression, it was demonstrated that ß2­adrenergic receptor (ADRB2) was the specific ß­adrenergic receptor subtype responsible for catecholamine release. In addition, it was demonstrated that the induction of autophagy was a novel consequence of ß2­adrenergic activation in GC cells. This was demonstrated by the appearance of double­membrane vesicles, punctuate GFP­RFP­microtubule­associated protein 1 light chain 3 distribution in the cytoplasm and a corresponding increase in autophagic flux. Notably, norepinephrine­induced autophagy was shown to have a tumor­promoting role under conditions of chronic stress in vitro and in vivo. It was further demonstrated that, upon activation of cAMP­response element binding protein, chronic stress promoted autophagic flux through the adenosine 5'­monophosphate­activated protein kinase­unc­51 like autophagy activating kinase 1 (AMPK­ULK1) pathway. Tissue microarray analysis revealed a negative correlation between the expression of ADRB2 and autophagic marker p62/sequestosome­1 in GC tumor samples. Additionally, high protein levels of ADRB2 correlated positively with tumor, node, metastasis stage and poor prognosis in patients with GC. These results establish a novel pathway that chronic stress activates tumor­promoting autophagy to accelerate the progression of GC. The present study is the first, to the best of our knowledge, providing preclinical evidence that chronic stress serves a role in the progression of GC.

Hsa_circRNA_103809 regulated the cell proliferation and migration in colorectal cancer via miR-532-3p / FOXO4 axis.

Circular RNAs(circRNAs) are a class of non-coding RNAs that are widely expressed in a variety of cell species. The role they play in cancers is poorly understood, especially in colorectal cancer (CRC). Hsa_circRNA_103809 (hsa_circ_0072088, circZFR)has been demonstrated to be lowly expressed in colorectal cancer tissues and is associated with stage and lymph node metastasis of cancer tissues. Real-time quantitative PCR (qRT-PCR) was used to verify the relationship of hsa_circRNA_103809 between colorectal cancer and paired adjacent tissue in clinical tissue samples. Then, the proliferative capacity, migration ability, cell cycle, and apoptosis were measured using wound-healing assay, CCK8, transwell assay, flow cytometry, and the like, when hsa_circRNA_103809 expression in SW620 and COCA-2. The qRT-PCR, western bolt and other experiments verify that the expression of hsa_circRNA_103809 can regulate the expression of miR-532-3P and FOXO4. Hsa_circRNA_103809 was found to be significantly down regulated in CRC tissues and cell lines and compared with paired adjacent non-tumorous tissues and normal FHC cells. Hsa_circRNA_103809 participates in the regulation of biological functions through the miR-532-3P/FOXO4 axis in the CRC. Hsa_circRNA_103809 may be a potential novel gene target for the diagnosis and treatment of CRC.

Clinical Application of Circulating Tumor DNA in the Genetic Analysis of Patients with Advanced GIST.

Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumor of digestive tract. In the past, tissue biopsy was the main method for the diagnosis of GISTs. Although, circulating tumor DNA (ctDNA) detection by next-generation sequencing (NGS) may be a feasible and replaceable method for diagnosis of GISTs. We retrospectively analyzed the data for ctDNA and tissue DNA detection from 32 advanced GIST patients. We found that NGS obviously increased the positive rate of ctDNA detection. ctDNA detection identified rare mutations that were not detected in tissue DNA detection. Tumor size and Ki-67 were significant influencing factors of the positive rate of ctDNA detection and concordance between ctDNA and tissue DNA detection. In all patients, the concordance rate between ctDNA and tissue DNA detection was 71.9%, with moderate concordance, but the concordance was strong for patients with tumor size > 10 cm or Ki-67 > 5%. Tumor size, mitotic figure, Ki-67, and ctDNA mutation type were the significant influencing factors of prognosis, but only tumor size and ctDNA mutation type, were the independent prognostic factors for advanced GIST patients. We confirmed that ctDNA detection by NGS is a feasible and promising method for the diagnosis and prognosis of advanced GIST patients. Mol Cancer Ther; 17(1); 290-6. ©2017 AACR.

miR-3174 Contributes to Apoptosis and Autophagic Cell Death Defects in Gastric Cancer Cells by Targeting ARHGAP10.

Gastric cancer (GC) is a major health problem worldwide because of its high morbidity and mortality. Considering the well-established roles of miRNA in the regulation of GC carcinogenesis and progression, we screened differentially expressed microRNAs (miRNAs) by using The Cancer Genome Atlas (TCGA) and the GEO databases. We found that miR-3174 was the most significantly differentially expressed miRNA in GC. Ectopic miR-3174 expression was also detected in clinical GC patient samples and cell lines and associated with poor patient prognosis. Apoptosis and autophagic cell death are two types of programmed cell death, whereas both are deficient in gastric cancer. Our functional analyses demonstrated that miR-3174 inhibited mitochondria-dependent apoptosis and autophagic cell death in GC. Moreover, high expression of miR-3174 also resulted in Cisplatin resistance in GC cells. Using bioinformatics analyses combined with in vitro and in vivo experiments, we determined that miR-3174 directly targets ARHGAP10. Notably, ARHGAP10 promoted mitochondria-dependent apoptosis by enhancing p53 expression, which was followed by Bax trans-activation and caspase cleavage. ARHGAP10 also facilitated autophagic cell death by suppressing mammalian target of rapamycin complex 1 (mTOC1) activity. Our results reveal a potential miRNA-based clinical therapeutic target that may also serve as a predictive marker for GC.

MicroRNA-148a-3p enhances cisplatin cytotoxicity in gastric cancer through mitochondrial fission induction and cyto-protective autophagy suppression.

Cisplatin (CDDP) resistance is a major clinical problem associated with poor prognosis in gastric cancer (GC) patients. In this study, we performed integrated analysis of TCGA data from microRNAs (miRNAs) expression matrix of GC patients who received CDDP-based chemotherapy with GEO dataset which contains differential miRNAs expression profiles in CDDP-resistant and -sensitive cell lines. We identified miR-148a-3p downregulation as a key step involved in CDDP resistance. Using a cohort consisting 105 GC patients who received CDDP-based therapy, we found that miR-148a-3p downregulation was associated with a decrease in patients' disease-free survival (DFS, P = 0.0077). A series of experiment data demonstrated that: 1) miR-148a-3p was downregulated in CDDP-resistant GC cell lines; 2) miR-148a-3p reconstitution sensitized CDDP-resistant cells to CDDP treatment through promoting mitochondrial fission and decreasing AKAP1 expression level; 3) AKAP1 played a novel role in CDDP resistance by inhibiting P53-mediated DRP1 dephosphorylation; 4) miR-148a-3p reconstitution in CDDP-resistant cells inhibits the cyto-protective autophagy by suppressing RAB12 expression and mTOR1 activation. Taken together, our study demonstrates that miR-148a-3p could be a promising prognostic marker or therapeutic candidate for overcoming CDDP resistance in GC.

Comprehensive Proteomic Characterization of the Human Colorectal Carcinoma Reveals Signature Proteins and Perturbed Pathways.

The global change in protein abundance in colorectal cancer (CRC) and its contribution to tumorigenesis have not been comprehensively analyzed. In this study, we conducted a comprehensive proteomic analysis of paired tumors and adjacent tissues (AT) using high-resolution Fourier-transform mass spectrometry and a novel algorithm of quantitative pathway analysis. 12380 proteins were identified and 740 proteins that presented a 4-fold change were considered a CRC proteomic signature. A significant pattern of changes in protein abundance was uncovered which consisted of an imbalance in protein abundance of inhibitory and activating regulators in key signal pathways, a significant elevation of proteins in chromatin modification, gene expression and DNA replication and damage repair, and a decreased expression of proteins responsible for core extracellular matrix architectures. Specifically, based on the relative abundance, we identified a panel of 11 proteins to distinguish CRC from AT. The protein that showed the greatest degree of overexpression in CRC compared to AT was Dipeptidase 1 (DPEP1). Knockdown of DPEP1 in SW480 and HCT116 cells significantly increased cell apoptosis and attenuated cell proliferation and invasion. Together, our results show one of largest dataset in CRC proteomic research and provide a molecular link from genomic abnormalities to the tumor phenotype.

SPOCK1 is up-regulated and promotes tumor growth via the PI3K/AKT signaling pathway in colorectal cancer.

SPOCK1 encodes a Ca2+-binding matricellular glycoprotein which plays an oncogenic role in cancer cells. However, the role of SPOCK1 in the pathogenesis of colorectal cancer (CRC) has not been determined. Here, SPOCK1 was found higher expressed in CRC tissues than that of adjacent normal tissues. Furthermore, up-regulated expression of SPOCK1 in CRC patients was associated with tumor size and TNM stage. In addition, we observed that the depletion of SPOCK1 inhibited proliferation in vitro and tumorigenicity in vivo and promoted apoptosis in cell culture. Our data suggest that inactivation of PI3K/Akt signaling pathway was involved in down-regulation of SPOCK1-mediated suppression of tumor cell proliferation. These results suggest that SPOCK1 expression is correlated with malignant features of CRC and may serve as potential therapeutic and preventive strategies for CRC.

Silencing speckle-type POZ protein by promoter hypermethylation decreases cell apoptosis through upregulating Hedgehog signaling pathway in colorectal cancer.

Epigenetic silencing of tumor suppressors contributes to the development and progression of colorectal cancer (CRC). We recently found that speckle-type POZ protein (SPOP) was significantly downregulated and the inactivation of SPOP promoted metastasis in CRC. This study aimed to clarify its epigenetic alteration, molecular mechanisms and clinical significance in CRC. Our results revealed that the core region of SPOP promoter was hypermethylated in CRC tissues and its methylation was correlated with poor survival. Transcription factor RXRA had a vital role in the regulation of SPOP gene. The data indicated that DNA methylation at -167 bp of the SPOP gene altered the binding affinity between transcription factor RXRA and SPOP promoter. Moreover, SPOP was found to associate with Gli2 and promoted its ubiquitination and degradation in CRC. Consequently, the expression level of Hh/Gli2 pathway-related apoptotic protein Bcl-2 was decreased and the function of resisting cell death was inhibited in CRC. It suggests that methylation status of SPOP promoter can be used as a novel epigenetic biomarker and a therapeutic target in CRC.