Single-cell analysis reveals the COL11A1+ fibroblasts are cancer-specific fibroblasts that promote tumor progression.

Background: Cancer-associated fibroblasts (CAFs) promote tumor progression through extracellular matrix (ECM) remodeling and extensive communication with other cells in tumor microenvironment. However, most CAF-targeting strategies failed in clinical trials due to the heterogeneity of CAFs. Hence, we aimed to identify the cluster of tumor-promoting CAFs, elucidate their function and determine their specific membrane markers to ensure precise targeting. Methods: We integrated multiple single-cell RNA sequencing (scRNA-seq) datasets across different tumors and adjacent normal tissues to identify the tumor-promoting CAF cluster. We analyzed the origin of these CAFs by pseudotime analysis, and tried to elucidate the function of these CAFs by gene regulatory network analysis and cell-cell communication analysis. We also performed cell-type deconvolution analysis to examine the association between the proportion of these CAFs and patients' prognosis in TCGA cancer cohorts, and validated that through IHC staining in clinical tumor tissues. In addition, we analyzed the membrane molecules in different fibroblast clusters, trying to identify the membrane molecules that were specifically expressed on these CAFs. Results: We found that COL11A1+ fibroblasts specifically exist in tumor tissues but not in normal tissues and named them cancer-specific fibroblasts (CSFs). We revealed that these CSFs were transformed from normal fibroblasts. CSFs represented a more activated CAF cluster and may promote tumor progression through the regulation on ECM remodeling and antitumor immune responses. High CSF proportion was associated with poor prognosis in bladder cancer (BCa) and lung adenocarcinoma (LUAD), and IHC staining of COL11A1 confirmed their specific expression in tumor stroma in clinical BCa samples. We also identified that CSFs specifically express the membrane molecules LRRC15, ITGA11, SPHK1 and FAP, which could distinguish CSFs from other fibroblasts. Conclusion: We identified that CSFs is a tumor specific cluster of fibroblasts, which are in active state, may promote tumor progression through the regulation on ECM remodeling and antitumor immune responses. Membrane molecules LRRC15, ITGA11, SPHK1 and FAP could be used as therapeutic targets for CSF-targeting cancer treatment.

MiR-585-5p impedes gastric cancer proliferation and metastasis by orchestrating the interactions among CREB1, MAPK1 and MITF.

Background: Gastric cancer (GC) is one of the most malignant and lethal cancers worldwide. Multiple microRNAs (miRNAs) have been identified as key regulators in the progression of GC. However, the underlying pathogenesis that miRNAs govern GC malignancy remains uncertain. Here, we identified a novel miR-585-5p as a key regulator in GC development. Methods: The expression of miR-585-5p in the context of GC tissue was detected by in situ hybridization for GC tissue microarray and assessed by H-scoring. The gain- and loss-of-function analyses comprised of Cell Counting Kit-8 assay and Transwell invasion and migration assay. The expression of downstream microphthalmia-associated transcription factor (MITF), cyclic AMP-responsive element-binding protein 1 (CREB1) and mitogen-activated protein kinase 1 (MAPK1) were examined by Immunohistochemistry, quantitative real-time PCR and western blot. The direct regulation between miR-585-5p and MITF/CREB1/MAPK1 were predicted by bioinformatic analysis and screened by luciferase reporter assay. The direct transcriptional activation of CREB1 on MITF was verified by luciferase reporter assay, chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays (EMSAs). The interaction between MAPK1 and MITF was confirmed by co-immunoprecipitation (Co-IP) and immunofluorescent double-labelled staining. Results: MiR-585-5p is progressively downregulated in GC tissues and low miR-585-5p levels were strongly associated with poor clinical outcomes. Further gain- and loss-of-function analyses showed that miR-585-5p possesses strong anti-proliferative and anti-metastatic capacities in GC. Follow-up studies indicated that miR-585-5p targets the downstream molecules CREB1 and MAPK1 to regulate the transcriptional and post-translational regulation of MITF, respectively, thus controlling its expression and cancer-promoting activity. MiR-585-5p directly and negatively regulates MITF together with CREB1 and MAPK1. According to bioinformatic analysis, promotor reporter gene assays, ChIP and EMSAs, CREB1 binds to the promotor region to enhance transcriptional expression of MITF. Co-IP and immunofluorescent double-labelled staining confirmed interaction between MAPK1 and MITF. Protein immunoprecipitation revealed that MAPK1 enhances MITF activity via phosphorylation (Ser73). MiR-585-5p can not only inhibit MITF expression directly, but also hinder MITF expression and pro-cancerous activity in a CREB1-/MAPK1-dependent manner indirectly. Conclusions: In conclusion, this study uncovered miR-585-5p impedes gastric cancer proliferation and metastasis by orchestrating the interactions among CREB1, MAPK1 and MITF.

Self-Assembly of Small Organic Molecules into Luminophores for Cancer Theranostic Applications.

Self-assembled biomaterials have been widely explored for real-time fluorescence imaging, imaging-guided surgery, and targeted therapy for tumors, etc. In particular, small molecule-based self-assembly has been established as a reliable strategy for cancer theranostics due to the merits of small-sized molecules, multiple functions, and ease of synthesis and modification. In this review, we first briefly introduce the supramolecular chemistry of small organic molecules in cancer theranostics. Then, we summarize and discuss advanced small molecule-based self-assembly for cancer theranostics based on three types, including peptides, amphiphilic molecules, and aggregation-induced emission luminogens. Finally, we conclude with a perspective on future developments of small molecule-based self-assembled biomaterials integrating diagnosis and therapy for biomedical applications. These applications highlight the opportunities arising from the rational design of small organic molecules with self-assembly properties for precision medicine.

Targeting the miR-34a/LRPPRC/MDR1 axis collapse the chemoresistance in P53 inactive colorectal cancer.

P53 mutation is an important cause of chemoresistance in colorectal cancer (CRC). The investigation and identification of the downstream targets and underlying molecular mechanism of chemoresistance induced by P53 abnormalities are therefore of great clinical significance. In this study, we demonstrated and reported for the first time that leucine-rich pentatricopeptide repeat-containing protein (LRPPRC) is a key functional downstream factor and therapeutic target for P53 mutation-induced chemoresistance. Due to its RNA binding function, LRPPRC specifically bound to the mRNA of multidrug resistance 1 (MDR1), increasing MDR1 mRNA stability and protein expression. In normal cells, P53 induced by chemotherapy inhibited the expression of LRPPRC via miR-34a and in turn reduced the expression of MDR1. However, chemotherapy-induced P53/miR-34a/LRPPRC/MDR1 signalling pathway activation was lost when P53 was mutated. Additionally, the accumulated LRPPRC and MDR1 promoted drug resistance. Most importantly, gossypol-acetic acid (GAA) was recently reported by our team as the first specific inhibitor of LRPPRC. In CRC cells with P53 mutation, GAA effectively induced degradation of the LRPPRC protein and reduced chemoresistance. Both in vivo and in vitro experiments revealed that combination chemotherapy with GAA and 5-fluorouracil (5FU) yielded improved treatment outcomes. In this study, we reported a novel mechanism and target related to P53-induced drug resistance and provided corresponding interventional strategies for the precision treatment of CRC.

Notch-mediated lactate metabolism regulates MDSC development through the Hes1/MCT2/c-Jun axis.

Myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) play critical roles in tumorigenesis. However, the mechanisms underlying MDSC and TAM development and function remain unclear. In this study, we find that myeloid-specific activation of Notch/RBP-J signaling downregulates lactate transporter MCT2 transcription via its downstream molecule Hes1, leading to reduced intracellular lactate levels, blunted granulocytic MDSC (G-MDSC) differentiation, and enhanced TAM maturation. We identify c-Jun as a novel intracellular sensor of lactate in myeloid cells using liquid-chromatography-mass spectrometry (LC-MS) followed by CRISPR-Cas9-mediated gene disruption. Meanwhile, lactate interacts with c-Jun to protect from FBW7 ubiquitin-ligase-mediated degradation. Activation of Notch signaling and blockade of lactate import repress tumor progression by remodeling myeloid development. Consistently, the relationship between the Notch-MCT2/lactate-c-Jun axis in myeloid cells and tumorigenesis is also confirmed in clinical lung cancer biopsies. Taken together, our current study shows that lactate metabolism regulated by activated Notch signaling might participate in MDSC differentiation and TAM maturation.

An MD2-perturbing peptide has therapeutic effects in rodent and rhesus monkey models of stroke.

Studies have failed to translate more than 1000 experimental treatments from bench to bedside, leaving stroke as the second leading cause of death in the world. Thrombolysis within 4.5 hours is the recommended therapy for stroke and cannot be performed until neuroimaging is used to distinguish ischemic stroke from hemorrhagic stroke. Therefore, finding a common and critical therapeutic target for both ischemic and hemorrhagic stroke is appealing. Here, we report that the expression of myeloid differentiation protein 2 (MD2), which is traditionally regarded to be expressed only in microglia in the normal brain, was markedly increased in cortical neurons after stroke. We synthesized a small peptide, Trans-trans-activating (Tat)-cold-inducible RNA binding protein (Tat-CIRP), which perturbed the function of MD2 and strongly protected neurons against excitotoxic injury in vitro. In addition, systemic administration of Tat-CIRP or genetic deletion of MD2 induced robust neuroprotection against ischemic and hemorrhagic stroke in mice. Tat-CIRP reduced the brain infarct volume and preserved neurological function in rhesus monkeys 30 days after ischemic stroke. Tat-CIRP efficiently crossed the blood-brain barrier and showed a wide therapeutic index for stroke because no toxicity was detected when high doses were administered to the mice. Furthermore, we demonstrated that MD2 elicited neuronal apoptosis and necroptosis via a TLR4-independent, Sam68-related cascade. In summary, Tat-CIRP provides robust neuroprotection against stroke in rodents and gyrencephalic nonhuman primates. Further efforts should be made to translate these findings to treat both ischemic and hemorrhagic stroke in patients.

Microsurgical Skills of Establishing Permanent Jugular Vein Cannulation in Rats for Serial Blood Sampling of Orally Administered Drug.

Blood sampling in small laboratory animals is necessary for pharmaceutical lead optimization but can cause great harm and stress to experimental animals, which could potentially affect results. The jugular vein cannulation (JVC) in rats is a widely used model for repeated blood collection but requires adequate training of surgery skills and animal care. This article details the microsurgical procedures for establishing and maintaining a permanent JVC rat model with specific focus on the placement and sealing of the jugular cannula. The importance of monitoring physiological (e.g., body weight, food, and water intake) and hematological parameters, was highlighted with results presented for 6 days post-surgery during the rat's recovery. The drug-plasma concentration-time profile of orally administered natural phenol ellagic acid was determined in the JVC rat model.

Regulation of the small GTPase Ran by miR-802 modulates proliferation and metastasis in colorectal cancer cells.

BACKGROUND: The small GTPase Ran is upregulated in multiple cancers and fundamental for cancer cell survival and progression, but its significance and molecular mechanisms in colorectal cancer (CRC) remain elusive. METHODS: Ran expression was detected in CRC cell lines and tumour tissues. In vitro and in vivo functional assays were performed to examine the effects of Ran on cell proliferation and metastasis. The pathways and effectors regulated by Ran were explored by an unbiased screening. Bioinformatics prediction and experimental validation were used to identify the miRNA regulator for Ran. RESULTS: Ran expression was frequently increased in metastatic CRC cells and tissues, especially in metastatic tissues. The upregulation of Ran correlated with poor CRC patient prognosis. Ran silencing reduced proliferation and metastasis of CRC cells both in vitro and in vivo. Ran regulated the expression of EGFR and activation of ERK and AKT signalling pathways. miR-802 was identified as an upstream regulator of Ran and miR-802 overexpression resulted in antiproliferative and antimetastatic activities. CONCLUSION: Our study demonstrates the oncogenic roles and underlying mechanisms of Ran in CRC and the novel miR-802/Ran/EGFR regulatory axis may provide potential biomarkers for the treatment of CRC.

A Phase I Trial of Berberine in Chinese with Ulcerative Colitis.

The Chinese natural product, berberine, has biological properties that support its potential efficacy as a colon cancer prevention agent. Its longstanding use in China to treat gastrointestinal tract and rheumatologic disorders is generally regarded as safe, supporting initial investigations in an at-risk population, such as individuals with ulcerative colitis. However, the safety of berberine in this population is not established. Individuals living in China with biopsy-proven ulcerative colitis, ≤grade 2 dysplasia, and with a ulcerative colitis disease activity index (UCDAI) score ≤1 on mesalamine, were randomized 3:1 in a double-blind phase I trial to berberine 900 mg/day or placebo for 3 months, with the primary objective of assessing safety. Blood samples and biopsies of the colorectum, from prespecified locations, were collected prior to and following therapy. Secondary endpoints included changes in UCDAI score, and in tissue and plasma markers of inflammation. Of toxicities at least possibly related, one episode of grade 3 elevation in transaminases and one episode of grade 1 nausea were observed among 12 individuals on berberine, and none were observed among 4 on placebo. The mean plasma berberine concentration was 3.5 nmol/L after berberine treatment, significantly higher than 0.5 nmol/L with placebo. Berberine significantly decreased the Geboes grade in colonic tissue, but had a nonsignificant effect on other tissue or blood biomarkers related to cell growth and inflammation. The combination of berberine and mesalamine is well tolerated in Chinese with ulcerative colitis and may enhance mesalamine's anti-inflammatory effects in colonic tissue.

Forkhead box K2 promotes human colorectal cancer metastasis by upregulating ZEB1 and EGFR.

Background: Metastasis is the major reason for high recurrence rates and poor survival among patients with colorectal cancer (CRC). However, the underlying molecular mechanism of CRC metastasis is unclear. This study aimed to investigate the role of forkhead box K2 (FOXK2), one of the most markedly increased FOX genes in CRC, and the mechanism by which it is deregulated in CRC metastasis. Methods: FOXK2 levels were analyzed in two independent human CRC cohorts (cohort I, n = 363; cohort II, n = 390). In vitro Transwell assays and in vivo lung and liver metastasis models were used to examine CRC cell migration, invasion and metastasis. Chromatin immunoprecipitation and luciferase reporter assays were used to measure the binding of transcription factors to the promoters of FOXK2, zinc finger E-box binding homeobox 1 (ZEB1) and epidermal growth factor receptor (EGFR). Cetuximab was utilized to treat FOXK2-mediated metastatic CRC. Results: FOXK2 was significantly upregulated in human CRC tissues, was correlated with more aggressive features and indicated a poor prognosis. FOXK2 overexpression promoted CRC migration, invasion and metastasis, while FOXK2 downregulation had the opposite effects. ZEB1 and EGFR were determined to be direct transcriptional targets of FOXK2 and were essential for FOXK2-mediated CRC metastasis. Moreover, activation of EGFR signaling by EGF enhanced FOXK2 expression via the extracellular regulated protein kinase (ERK) and nuclear factor (NF)-κB pathways. The EGFR monoclonal antibody cetuximab significantly inhibited FOXK2-promoted CRC metastasis. In clinical CRC tissues, FOXK2 expression was positively correlated with the expression of p65, ZEB1 and EGFR. CRC patients who coexpressed p65/FOXK2, FOXK2/ZEB1 and FOXK2/EGFR had poorer prognosis. Conclusions: FOXK2 serves as a prognostic biomarker in CRC. Cetuximab can block the EGF-NF-κB-FOXK2-EGFR feedback loop and suppress CRC metastasis.

COL4A2 in the tissue-specific extracellular matrix plays important role on osteogenic differentiation of periodontal ligament stem cells.

Periodontal ligament stem cells (PDLSCs) can repair alveolar bone defects in periodontitis in a microenvironment context-dependent manner. This study aimed to determine whether different extracellular matrices (ECMs) exert diverse effects on osteogenic differentiation of PDLSCs and accurately control alveolar bone defect repair. Methods: The characteristics of PDLSCs and bone marrow mesenchymal stem cells (BMSCs) with respect to surface markers and multi-differentiation ability were determined. Then, we prepared periodontal ligament cells (PDLCs)-derived and bone marrow cells (BMCs)-derived ECMs (P-ECM and B-ECM) and the related decellularized ECMs (dECMs). Transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM), and protein mass spectrometry were used to distinguish the ECMs. The expression of Type IV collagen A2 (COL4A2) in the ECMs was inhibited by siRNA or activated by lentiviral transduction of relevant cells. The stemness, proliferation, and differentiation of PDLSCs were determined in vitro in different dECMs. For the in vivo analysis, different dECMs under the regulation of COL4A2 mixed with PDLSCs and Bio-Oss bone powder were subcutaneously implanted into immunocompromised mice or in defects in rat alveolar bone. The repair effects were identified by histological or immunohistochemical staining and micro-CT. Results: B-dECM exhibited more compact fibers than P-dECM, as revealed by TEM, SEM, and AFM. Protein mass spectrometry showed that COL4A2 was significantly increased in B-dECM compared with P-dECM. PDLSCs displayed stronger proliferation, stemness, and osteogenic differentiation ability when cultured on B-dECM than P-dECM. Interestingly, B-dECM enhanced the osteogenic differentiation of PDLSCs to a greater extent than P-dECM both in vitro and in vivo, whereas downregulation of COL4A2 in B-dECM showed the opposite results. Furthermore, the classical Wnt/ß-catenin pathway was found to play an important role in the negative regulation of osteogenesis through COL4A2, confirmed by experiments with the Wnt inhibitor DKK-1 and the Wnt activator Wnt3a. Conclusion: These findings indicate that COL4A2 in the ECM promotes osteogenic differentiation of PDLSCs through negative regulation of the Wnt/ß-catenin pathway, which can be used as a potential therapeutic strategy to repair bone defects.

Correction: O-GlcNAcylation promotes colorectal cancer metastasis via the miR-101-O-GlcNAc/EZH2 regulatory feedback circuit.

A correction to this paper has been published and can be accessed via a link at the top of the paper.

OGDH mediates the inhibition of SIRT5 on cell proliferation and migration of gastric cancer.

SIRT5 has a wide range of functions in different cellular processes such as glycolysis, TCA cycle and antioxidant defense, which mediates lysine desuccinylation, deglutarylation and demalonylation. Recent evidences have implicated that SIRT5 is a potential suppressor of gastric cancer (GC). However, the underlying mechanism of SIRT5 in gastric cancer is still unclear. Here, we show that SIRT5 expression is significantly decreased in human GC tissues. Functional analysis demonstrates that SIRT5 inhibits cell growth in vitro and in vivo, arrests the cell cycle in G1/S transition, and suppresses migration and invasion of GC cells via regulating epithelial-to-mesenchymal transition. Mechanistically, we demonstrate that there is the direct interaction between SIRT5 and 2-oxoglutarate dehydrogenase (OGDH), and desuccinylation of OGDH by SIRT5 inhibits the activity of OGDH complex. Further studies of the relationship between SIRT5 and OGDH show OGDH inhibition by succinyl phosphonate (SP) or siRNA suppresses the increase in cell growth and migration induced by SIRT5 deletion. Moreover, SIRT5 decreases mitochondrial membrane potential (ΔΨm), ATP products and increases the ROS levels and NADP/NADPH ratio in GC cells through the inhibition of OGDH complex activity. Therefore, SIRT5 suppresses GC cell growth and migration through desuccinylating OGDH and inhibiting OGDH complex activity to disturb mitochondrial functions and redox status.

O-GlcNAcylation promotes colorectal cancer metastasis via the miR-101-O-GlcNAc/EZH2 regulatory feedback circuit.

Advanced colorectal cancer (CRC) is one of the deadliest cancers, and the 5-year survival rate of patients with metastasis is extremely low. The epithelial-mesenchymal transition (EMT) is considered essential for metastatic CRC, but the fundamental molecular basis underlying this effect remains unknown. Here, we identified that O-GlcNAcylation, a unique posttranslational modification (PTM) involved in cancer metabolic reprogramming, increased the metastatic capability of CRC. The levels of O-GlcNAcylation were increased in the metastatic CRC tissues and cell lines, which likely promoted the EMT by enhancing EZH2 protein stability and function. The CRC patients with higher levels of O-GlcNAcylation exhibited greater lymph node metastasis potential and lower overall survival. Bioinformatic analysis and luciferase reporter assays revealed that both O-GlcNAcylation transferase (OGT) and EZH2 are posttranscriptionally inhibited by microRNA-101. In addition, O-GlcNAcylation and H3K27me3 modification in the miR-101 promoter region further inhibited the transcription of miR-101, resulting in the upregulation of OGT and EZH2 in metastatic CRC, thus forming a vicious cycle. In this study, we demonstrated that O-GlcNAcylation, which is negatively regulated by microRNA-101, likely promotes CRC metastasis by enhancing EZH2 protein stability and function. Reducing O-GlcNAcylation may be a potential therapeutic strategy for metastatic CRC.

Novel peptide GX1 inhibits angiogenesis by specifically binding to transglutaminase-2 in the tumorous endothelial cells of gastric cancer.

The clinical application of GX1, an optimal gastric cancer (GC) targeting peptide, is greatly limited because its receptor in the GC vasculature is unknown. In this study, we screened the candidate receptor of GX1, transglutaminase-2(TGM2), by co-immunoprecipitation (co-IP) combined with mass spectrometry. We found that TGM2 was up-regulated in GC vascular endothelial cells and that GX1 receptor expression was suppressed correspondingly after TGM2 downregulation. A highly consistent co-localization of GX1 receptor and TGM2 was detected at both the cellular and tissue levels. High TGM2 expression was evident in GC tissues from patients with poor prognosis. After TGM2 downregulation, the GX1-mediated inhibition of proliferation and migration and the induction of the apoptosis of GC vascular endothelial cells were weakened or even reversed. Finally, we observed that GX1 could inhibit the GTP-binding activity of TGM2 by reducing its intracellular distribution and downregulating its downstream molecular targets (nuclear factor-kappa B, NF-κB; hypoxia-inducible factor 1-α, HIF1α) in GC vascular endothelial cells. Our study confirms that peptide GX1 can inhibit angiogenesis by directly binding to TGM2, subsequently reducing the GTP-binding activity of TGM2 and thereby suppressing its downstream pathway(NF-κB/HIF1α). Our conclusions suggest that GX1/TGM2 may provide a new target for the diagnosis and treatment of GC.

Combined cell surface carbonic anhydrase 9 and CD147 antigens enable high-efficiency capture of circulating tumor cells in clear cell renal cell carcinoma patients.

Circulating tumor cells (CTCs) have emerged as promising tools for noninvasive cancer detection and prognosis. Most conventional approaches for capturing CTCs use an EpCAM-based enrichment strategy, which does not work well in cancers that show low or no expression of EpCAM, such as renal cell carcinoma (RCC). In this study, we developed a new set of cell surface markers including CA9 and CD147 as alternative CTC-capture antigens specifically designed for RCC patients. We showed that the expression of both CA9 and CD147 was prevalent in a RCC patient cohort (n=70) by immunohistochemical analysis, with both molecules in combination covering 97.1% of cases. The NanoVelcro platform combined with CA9-/CD147-capture antibodies demonstrated significantly higher efficiency for capturing both CTC-mimicking renal cancer cells and RCC CTCs in peripheral blood, compared to the conventional EpCAM-based method. Using immunofluorescence cytological validation at the single-cell level, we were able to identify bona fide CTCs in RCC patient blood following the well-accepted criteria in our CTC-capture system. We further demonstrated a significant association of CTC numbers as well as the CTC expression status of Vimentin, a mesenchymal marker, with disease progression, including pathologic features and clinical staging. These results provide new insights into developing novel, effective targets/approaches for capturing CTCs, making CTCs a valuable tool for improved cancer detection, prognosis and treatment in RCC.

A possible role of GDDR in the development of Helicobacter pylori-associated gastric cancer.

Helicobacter pylori (H. pylori) infection plays an important part in the development of gastric carcinoma. GDDR has been confirmed as a tumor suppressor gene in gastric tumorigenesis. However, the underlying mechanism of GDDR in H. pylori-induced carcinogenesis is not well known. The aim of this study is to investigate the clinicopathological significance and possible molecular mechanism of GDDR in gastric cancer associated with H. pylori. Western blot, real-time quantitative PCR (qRT-PCR), and immunohistochemistry were used to detect the expression level of GDDR with or without H. pylori infection. The function and possible related molecular mechanisms of GDDR were further explored in vitro and in vivo. The variability of GDDR expression appeared in the early stage of gastric carcinogenesis with positive H. pylori infection status. GDDR might inhibit the progression of normal gastric epithelial cells to cancer cells by suppressing NF-kappaB signaling pathway, which in turn could be regulated by H. pylori infection. Our results suggested, for the first time, that the gradual change in GDDR expression might not only be directly related to H. pylori infection but also be an early molecular event in the development of gastric carcinoma.

Association of TLR4 and Treg in Helicobacter pylori Colonization and Inflammation in Mice.

The host immune response plays an important role in the pathogenesis of Helicobacter pylori infection. The aim of this study was to clarify the immune pathogenic mechanism of Helicobacter pylori infection via TLR signaling and gastric mucosal Treg cells in mice. To discover the underlying mechanism, we selectively blocked the TLR signaling pathway and subpopulations of regulatory T cells in the gastric mucosa of mice, and examined the consequences on H. pylori infection and inflammatory response as measured by MyD88, NF-κB p65, and Foxp3 protein expression levels and the levels of Th1, Th17 and Th2 cytokines in the gastric mucosa. We determined that blocking TLR4 signaling in H. pylori infected mice decreased the numbers of Th1 and Th17 Treg cells compared to controls (P < 0.001-0.05), depressed the immune response as measured by inflammatory grade (P < 0.05), and enhanced H. pylori colonization (P < 0.05). In contrast, blocking CD25 had the opposite effects, wherein the Th1 and Th17 cell numbers were increased (P < 0.001-0.05), immune response was enhanced (P < 0.05), and H. pylori colonization was inhibited (P < 0.05) compared to the non-blocked group. In both blocked groups, the Th2 cytokine IL-4 remained unchanged, although IL-10 in the CD25 blocked group was significantly decreased (P < 0.05). Furthermore, MyD88, NF-κB p65, and Foxp3 in the non-blocked group were significantly lower than those in the TLR4 blocked group (P < 0.05), but significantly higher than those of the CD25 blocked group (P < 0.05). Together, these results suggest that there might be an interaction between TLR signaling and Treg cells that is important for limiting H. pylori colonization and suppressing the inflammatory response of infected mice.

Chaperone-mediated autophagy regulates proliferation by targeting RND3 in gastric cancer.

LAMP2A is the key protein of chaperone-mediated autophagy (CMA), downregulation of LAMP2A leads to CMA blockade. CMA activation has been implicated in cancer growth, but the exact mechanisms are unclear. Elevated expression of LAMP2A was found in 8 kinds of tumors (n=747), suggesting that LAMP2A may have an important role in cancer progression. Unsurprisingly, LAMP2A knockdown in gastric cancer (GC) cells hindered proliferation, accompanied with altered expression of cell cycle-related proteins and accumulation of RND3/RhoE. Interactomic and KEGG analysis revealed that RND3 was a putative CMA substrate. Further study demonstrated that RND3 silencing could partly rescue the proliferation arrest induced by LAMP2A knockdown; RND3 was increased upon lysosome inhibition via both chemicals and LAMP2A-shRNA; Furthermore, RND3 could interact with CMA components HSPA8 and LAMP2A, and be engulfed by isolated lysosomes. Thus, constant degradation of RND3 by CMA is required to sustain rapid proliferation of GC cells. At last, the clinical significance of LAMP2A was explored in 593 gastric noncancerous lesions and 173 GC tissues, the results revealed that LAMP2A is a promising biomarker for GC early warning and prognosis of female GC patients.

POU2F2-oriented network promotes human gastric cancer metastasis.

BACKGROUND AND AIMS: Aberrant upregulation of POU2F2 expression has been discovered in metastatic gastric cancer (GC). However, the mechanisms underlying the aberrant upregulation and the potential functions of POU2F2 remain uncertain. DESIGN: The role and mechanism of POU2F2 in GC metastasis were investigated in gastric epithelial cells, GC cell lines and an experimental metastasis animal model by gain of function and loss of function. Upstream and downstream targets of POU2F2 were selected by bioinformatics and identified by luciferase reporter assay, electrophoretic mobility shift assay and chromatin immunoprecipitation PCR. The influence of miR-218 on its putative target genes (POU2F2, ROBO1 and IKK-ß) and GC metastasis was further explored via in vitro and in vivo approaches. RESULTS: Increased POU2F2 expression was detected in metastatic GC cell lines and patient samples. POU2F2 was induced by the activation of nuclear factor (NF)-κB and, in turn, regulated ROBO1 transcription, thus functionally contributing to GC metastasis. Finally, miR-218 was found to suppress GC metastasis by simultaneously mediating multiple molecules in the POU2F2-oriented network. CONCLUSIONS: This study demonstrated that NF-κB and the SLIT2/ROBO1 interaction network with POU2F2 as the central part may exert critical effects on tumour metastasis. Blocking the activation of the POU2F2-oriented metastasis network using miR-218 precursors exemplified a promising approach that sheds light on new strategies for GC treatment.