Macrophage exosomes mediate palmitic acid-induced metainflammation by transferring miR-3064-5p to target IκBα and activate NF-κB signaling.

INTRODUCTION: High palmitic acid (PA) levels trigger metainflammation, facilitating the onset and progression of chronic metabolic diseases. Recently, exosomes were identified as new inflammation mediators. However, the mechanism by which macrophage exosomes mediate PA-induced inflammation remains unclear. OBJECTIVES: To explore how PA induces metainflammation through macrophage exosomes. METHODS: Exosomes secreted by RAW264.7 mouse macrophages stimulated with PA (ExosPA) or not (Exos) were prepared by ultracentrifugation. The differential miRNAs between ExosPA and Exos were identified by high-throughput sequencing, and their targeted mRNAs and proteins were bioinformatically analyzed and verified by qPCR and western blot. Mouse macrophages and metabolic cells (AML-12 hepatocytes, C2C12 myocytes or 3T3-L1 adipocytes) were treated with ExosPA or Exos. The verified miRNAs and its targeted molecules related to inflammation were analyzed in recipient cells. Furthers, exosomes were prepared from primary peritoneal macrophages isolated from AIN93G diet-fed (Control PM-Exos) or HPD-fed (PA PM-Exos) mice. Control or PA PM-Exos were then tail vein injected (30 µg) into mice (n = 10), once a week for 2 weeks. The verified miRNA and its targets in blood, blood exosomes, and metabolic tissues were detected. Finally, measured the levels of miRNA, inflammatory factors, and fatty acids in the blood of 20 obese/overweight individuals and 20 healthy individuals. RESULTS: ExoPA activate NF-κB signaling and enhance inflammatory enzyme/cytokine production in macrophages and metabolic cells. ExoPA enrich miR-3064-5p and target to inhibit IκBα as verified by exosome inhibitors and miR-3064-5p mimics and inhibitors. HPD elevates exosomal miR-3064-5p, macrophage exosomal miR-3064-5p, and inflammatory cytokine levels in mice circulation. PA PM-Exos from HPD-fed mice triggered inflammation in the circulation and metabolic tissues/organs of chow diet-fed mice. Overweight/obese individuals exhibit increased levels of circulating palmitoleic acid, exosomal miR-3064-5p, and high-sensitivity C-reactive proteins. CONCLUSIONS: Macrophage exosomes transferring miR-3064-5p to target IκBα and activate NF-κB signaling in metabolic cells is a mechanism of PA-induced metainflammation.

Gut Microbiota Plays Essential Roles in Soyasaponin's Preventive Bioactivities against Steatohepatitis in the Methionine and Choline Deficient (MCD) Diet-Induced Non-Alcoholic Steatohepatitis (NASH) Mice.

SCOPE: Gut microbiota (GM) is involved in nonalcoholic steatohepatitis (NASH) development. Phytochemicals soyasaponins can prevent NASH possibly by modulating GM. This study aims to investigate the preventive bioactivities of soyasaponin monomers (SS-A1 and SS-Bb) against NASH and explores the mechanisms by targeting GM. METHODS AND RESULTS: Male C57BL/6 mice are fed with methionine and choline deficient (MCD) diet containing SS-A1 , SS-Bb, or not for 16 weeks. Antibiotics-treated pseudo germ-free (PGF) mice are fed with MCD diet containing SS-A1 , SS-Bb, or not for 8 weeks. GM is determined by 16S rRNA amplicon sequencing. Bile acids (BAs) are measured by UPLC-MS/MS. In NASH mice, SS-A1 and SS-Bb alleviate steatohepatitis and fibrosis, reduce ALT, AST, and LPS in serum, decrease TNF-α, IL-6, α-SMA, triglycerides, and cholesterol in liver. SS-A1 and SS-Bb decrease Firmicutes, Erysipelotrichaceae, unidentified-Clostridiales, Eggerthellaceae, Atopobiaceae, Aerococcus, Jeotgalicoccus, Gemella, Rikenella, increase Proteobacteria, Verrucomicrobia, Akkermansiaceae, Romboutsia, and Roseburia. SS-A1 and SS-Bb alter BAs composition in liver, serum, and feces, activate farnesoid X receptor (FXR) in liver and ileum, increase occludin and ZO-1 in intestine. However, GM clearance abrogates the preventive bioactivities of SS-A1 and SS-Bb against NASH. CONCLUSION: GM plays essential roles in soyasaponin's preventive bioactivities against steatohepatitis in MCD diet-induced NASH mice.

Next-generation sequencing identifies CDKN2A alterations as prognostic biomarkers in recurrent or metastatic head and neck squamous cell carcinoma predominantly receiving immune checkpoint inhibitors.

Background: This study aimed to identify potential biomarkers in patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) and further probe the prognostic implications of CDKN2A mutations, particularly within a subset receiving immunotherapy. Methods: In this retrospective single-center study, we evaluated the next-generation sequencing (NGS) data from Foundation Medicine (FM) for patients with recurrent or metastatic HNSCC between January 1, 2019, and December 31, 2021. Patients were stratified based on CDKN2A loss-of-function (LOF) versus wild-type (WT) categorizations, with a focused subgroup analysis on those administered immunotherapy. Results: The study encompassed 77 patients, of which 62 had undergone immunotherapy. The median duration of follow-up was 22.6 months. For the CDKN2A LOF group, the median overall survival (OS) was 16.5 months, contrasted with 30.0 months in the CDKN2A WT group (P=0.014). Notably, female gender (hazard ratio [HR]=4.526, 95% confidence interval [CI]: 1.934-10.180, P=0.0003) and CDKN2A LOF (HR=2.311, 95% CI: 1.156-4.748, P=0.019) emerged as independent risk factors for mortality in patients with recurrent or metastatic HNSCC. Within the immunotherapy subset, the median OS was 11.7 months for the CDKN2A LOF group, and 22.5 months for the CDKN2A WT group (P=0.017). Further, the female gender (HR=4.022, 95% CI: 1.417-10.710, P=0.006), CDKN2A LOF (HR=4.389, 95% CI: 1.782-11.460, P=0.002), and a combined positive score below 1 (HR=17.20, 95% CI: 4.134-79.550, P<0.0001) were identified as significant predictors of mortality among patients with recurrent or metastatic HNSCC receiving immunotherapy. Conclusion: Alterations manifesting as LOF in the CDKN2A gene stand as robust indicators of unfavorable survival outcomes in HNSCC patients, including the subset that underwent immunotherapy.

Coordinated modulation of long non-coding RNA ASBEL and curcumin co-delivery through multicomponent nanocomplexes for synchronous triple-negative breast cancer theranostics.

BACKGROUND: Abnormally regulated long non-coding RNAs (lncRNAs) functions in cancer emphasize their potential to serve as potential targets for cancer therapeutic intervention. LncRNA ASBEL has been identified as oncogene and an anti-sense transcript of tumor-suppressor gene of BTG3 in triple-negative breast cancer (TNBC). RESULTS: Herein, multicomponent self-assembled polyelectrolyte nanocomplexes (CANPs) based on the polyelectrolytes of bioactive hyaluronic acid (HA) and chitosan hydrochloride (CS) were designed and prepared for the collaborative modulation of oncogenic lncRNA ASBEL with antago3, an oligonucleotide antagonist targeting lncRNA ASBEL and hydrophobic curcumin (Cur) co-delivery for synergetic TNBC therapy. Antago3 and Cur co-incorporated CANPs were achieved via a one-step assembling strategy with the cooperation of noncovalent electrostatic interactions, hydrogen-bonding, and hydrophobic interactions. Moreover, the multicomponent assembled CANPs were ulteriorly decorated with a near-infrared fluorescence (NIRF) Cy-5.5 dye (FCANPs) for synchronous NIRF imaging and therapy monitoring performance. Resultantly, MDA-MB-231 cells proliferation, migration, and invasion were efficiently inhibited, and the highest apoptosis ratio was induced by FCANPs with coordination patterns. At the molecular level, effective regulation of lncRNA ASBEL/BTG3 and synchronous regulation of Bcl-2 and c-Met pathways could be observed. CONCLUSION: As expected, systemic administration of FCANPs resulted in targeted and preferential accumulation of near-infrared fluorescence signal and Cur in the tumor tissue. More attractively, systemic FCANPs-mediated collaborative modulating lncRNA ASBEL/BTG3 and Cur co-delivery significantly suppressed the MDA-MB-231 xenograft tumor growth, inhibited metastasis and extended survival rate with negligible systemic toxicity. Our present study represented an effective approach to developing a promising theranostic platform for combating TNBC in a combined therapy pattern.

Milk Exosomes from Gestational Diabetes Mellitus (GDM) and Healthy Parturient Exhibit Differential miRNAs Profiles and Distinct Regulatory Bioactivity on Hepatocyte Proliferation.

SCOPE: Exosomes, a novel type of bioactive component in human milk (HM), affect infant development, growth, and health. Recent studies indicate that HM exosomes and miRNAs relate to gestational diabetes mellitus (GDM). However, the miRNAs profiles and functionalities of HM exosomes from GDM parturient remain unclear. This study aims to compare the differential miRNAs in HM exosomes from GDM and healthy parturient, and investigate the HM exosomes bioactivities in regulating hepatocyte proliferation and insulin sensitivity. METHODS AND RESULTS: This study extracted HM exosomes from GDM (GDM-EXO) and healthy (NOR-EXO) parturient by ultracentrifugation, high-throughput sequenced and compared the exosomal miRNAs profiles, and explored the regulatory bioactivities on hepatocyte proliferation in HepG2 cells and Balb/c mice. As compared to NOR-EXO, GDM-EXO has similar morphology, size, concentration, and exosome-specific markers (CD9 and TSG101) expression. GDM-EXO and NOR-EXO specifically harbor 1299 and 8 miRNAs, respectively. Moreover, GDM-EXO had 176 upregulated and 47 downregulated miRNAs compared with NOR-EXO. Both GDM-EXO and NOR-EXO were absorbed in cultured HepG2 hepatocytes and mice liver. GDM-EXO inhibited hepatocytes proliferation by downregulating mammalian target of rapamycin (mTOR) possibly via exosomal miR-101-3p delivery. CONCLUSION: HM exosomes from GDM and healthy parturient exhibit differential miRNAs profiles and distinct regulatory bioactivity on hepatocyte proliferation.

Role of ROR2 in promoting gastric cancer metastasis by enhancing c-JUN-mediated MMP3 transcription.

Background: Receptor tyrosine kinase-like orphan receptor 2 (ROR2) is a transmembrane receptor that has a complex role in cancer, acting either to promote or inhibit tumor progression in different tumor types. The effect of ROR2 on gastric cancer is unclear. Methods: Immunohistochemistry was used to investigate the role of ROR2 in the prognosis of gastric cancer. Transwell assay and a BALB/c nude mice pulmonary metastasis model were used to ascertain the role of ROR2 in promoting metastasis in vitro and in vivo. A protein expression array, chromatin immunoprecipitation (ChIP) assay, and luciferase reporter assay were employed to search for the target genes of ROR2. Results: ROR2 was found to be upregulated in gastric cancer tissues, which was correlated with poor disease-free survival (DFS) and overall survival (OS) in gastric cancer patients. Moreover, ROR2 promoted gastric cancer cell migratory and invasive behaviors in vitro and metastasis in vivo. Further research showed that ROR2 promoted gastric cancer metastasis via upregulation of matrix metalloproteinase 3 (MMP3). Analyses of clinical data indicated that high expression of ROR2 was correlated with a high expression of MMP3. Further study showed that ROR2 activated c-JUN by translocating phosphorylated JNK1/2 into the nucleus, and c-JUN interacted directly with the MMP3 promoter, leading to enhanced MMP3 transcription. Conclusions: We report for the first time that ROR2 is upregulated in gastric cancer, promotes metastasis, and is associated with poor prognosis in gastric cancer. The findings suggest that ROR2 may be a promising prognostic predictor for gastric cancer. Silencing the JNK1/2-c-JUN pathway, thereby inhibiting MMP3 expression, may serve as a promising strategy to inhibit gastric cancer progression.

Safety, tolerability, and pharmacokinetics/pharmacodynamics of JMT103 in patients with bone metastases from solid tumors.

Bone metastases are common complications of solid tumors. The outcome is poor despite major progress in cancer therapies. We describe a multicenter, open-label, phase 1, dose escalation and expansion trial of JMT103, a novel fully humanized receptor activator of nuclear factor kappa-B ligand (RANKL)-targeting monoclonal antibody, in adults with bone metastases from solid tumors. The study assessed the safety, tolerability, and pharmacokinetics/pharmacodynamics of JMT103. Patients received JMT103 at doses of 0.5, 1.0, 2.0, and 3.0 mg/kg every 4 weeks for 3 cycles. Among 59 patients enrolled, 20 and 39 patients participated in the dose-escalation and dose-expansion phases, respectively. One dose-limiting toxicity was observed at 2.0 mg/kg. The maximum tolerated dose was not determined. Treatment-related adverse events were reported in 29 (49.2%) patients, most commonly hypophosphatemia (30.5%), hypocalcemia (23.7%), and hypermagnesemia (10.2%). No treatment-related serious adverse events were reported. Two patients died due to disease progression, which were attributed to gastric cancer and lung neoplasm malignant respectively. Dose proportionality occurred between exposure levels and administered dose was within a dose range of 0.5 to 3.0 mg/kg. The suppression of urinary N-telopeptide corrected for creatinine was rapid, significant, and sustained across all doses of JMT103, with the median change from baseline ranging from -61.4% to -92.2% at day 141. JMT103 was well tolerated in patients with bone metastases from solid tumors, with a manageable safety profile. Bone antiresorptive activity shows the potential of JMT103 for treatment of bone metastases from solid tumors. Registration No.: NCT03550508; URL: https://www.clinicaltrials.gov/.

Targeting type Iγ phosphatidylinositol phosphate kinase overcomes oxaliplatin resistance in colorectal cancer.

Rationale: Oxaliplatin is a widely used chemotherapy drug for advanced colorectal cancer (CRC) and its resistance is a major challenge for disease treatment. However, the molecular mechanism underlying oxaliplatin resistance remains largely elusive. Methods: An integrative analysis was performed to determine differentially expressed genes involved in oxaliplatin resistance. Loss- and gain-of-function studies were employed to investigate the roles of type Iγ phosphatidylinositol phosphate kinase (PIPKIγ) on oxaliplatin resistance in CRC cells. Exosomes derived from CRC cell lines were assessed for PD-L1 level and the ability to promote oxaliplatin resistance. Quantitative real-time PCR, immunofluorescence, luciferase reporter assay, Western blotting and other techniques were conducted to decipher the molecular mechanism. Results: PIPKIγ was identified as a critical gene related to oxaliplatin resistance in CRC. Genetic manipulation studies revealed that PIPKIγ profoundly facilitated oxaliplatin resistance and affected the expression of DNA damage repair proteins. Mechanistically, PIPKIγ promoted the expression of the immune checkpoint molecule PD-L1 via activation of NF-κB signaling pathway. Genetic silencing of PD-L1 did not affect CRC cell proliferation but significantly sensitized CRC cells to oxaliplatin. Notably, PD-L1 was revealed to be encapsulated in the exosomes, and the addition of exosomal PD-L1 to sh-PD-L1 CRC cells restored oxaliplatin resistance. Pharmacological hijacking PIPKIγ-exosomal PD-L1 axis largely reduced oxaliplatin resistance in CRC cells. In vivo experiments showed that PD-L1 loss significantly blocked oxaliplatin resistance and the addition of PD-L1-enriched exosomes promoted tumor growth and reduced mouse survival time. Conclusion: Our findings reveal a previous unprecedented role of PIPKIγ in oxaliplatin resistance and provide a key mechanism of exosomal PD-L1 in CRC with potential therapeutics.

miR-335 targets CRIM1 to promote the proliferation and inhibit the apoptosis of placental trophoblast cells in preeclamptic rats.

OBJECTIVE: To investigate the role of miRNA-335 on the proliferation and apoptosis of placental trophoblast cells in preeclamptic rats and its potential mechanism. METHODS: Placental trophoblast cells were isolated from preeclamptic model rats and normal ones. Trophoblast cells from the model group were divided into six groups for transfection: the blank (empty vector) group, the NC (negative control) group, the miRNA-335 mimic group, the miRNA-335 inhibitor group, the CRIM1 (overexpressed recombinant plasmid) group, and the miRNA-335 inhibitor + CRIM1 group. The miRNA-335 expressions after the transfection were determined using qRT-PCR. The mRNA and protein expressions of CRIM1, the transforming growth factor (TGF-ß1), and the apoptosis-related factors (Bax and Bcl-2) in each group were determined using qRT-PCR and Western blotting. The cell proliferation and apoptosis were determined using MTT assays and flow cytometry, respectively. RESULTS: Compared with normal rats, the systolic blood pressure, diastolic blood pressure, and urinary protein levels were increased in the model rats, which had increased miRNA-335 expressions, but a decreased CRIM1 expressions (all P<0.05). The inhibition of miRNA-335 promoted the expressions of CRIM1, TGF-ß1, and Bcl-2 and inhibited the expression of Bax in trophoblast cells (all P<0.05). miRNA-335 inhibition or CRIM1 over-expression promoted the proliferation and reduced the apoptosis of trophoblast cells. The combined effect of miRNA-335 inhibition or CRIM1 over-expression had an even more significant effect on the changes in the above indicators (all P<0.05). CONCLUSION: miRNA-335 inhibition can enhance the expression of CRIM1 to promote the proliferation and reduce the apoptosis of trophoblast cells in preeclamptic rats.

Advancements in electrochemical biosensing for respiratory virus detection: A review.

Respiratory viruses are real menace for human health which result in devastating epidemic disease. Consequently, it is in urgent need of identifying and quantifying virus with a rapid, sensitive and precise approach. The study of electrochemical biosensors for respiratory virus detection has become one of the most rapidly developing scientific fields. Recent developments in electrochemical biosensors concerning respiratory virus detection are comprehensively reviewed in this paper. This review is structured along common detecting objects of respiratory viruses, electrochemical biosensors, electrochemical biosensors for respiratory virus detection and future challenges. The electrochemical biosensors for respiratory virus detection are introduced, including nucleic acids-based, immunosensors and other affinity biosensors. Lastly, for Coronavirus disease 2019 (COVID-19) diagnosis, the future challenges regarding developing electrochemical biosensor-based Point-of-Care Tests (POCTs) are summarized. This review is expected to provide a helpful guide for the researchers entering this interdisciplinary field and developing more novel electrochemical biosensors for respiratory virus detection.

Corrigendum to "PIPKIγ Regulates CCL2 Expression in Colorectal Cancer by Activating AKT-STAT3 Signaling".

[This corrects the article DOI: 10.1155/2019/3690561.].

PIPKIγ Regulates CCL2 Expression in Colorectal Cancer by Activating AKT-STAT3 Signaling.

Colorectal cancer (CRC) remains the third most commonly diagnosed cancer, ranking second among the most common causes of cancer-related mortality. Immune checkpoint therapy has recently been shown to have great potential. However, only some patients respond to immune checkpoint blockade, indicating the unmet need for determining the underlying mechanism of colorectal cancer immunosuppression. In this study, we analyzed The Cancer Genome Atlas (TCGA) datasets and found that high expression of PIPKIγ positively correlated with tumor-associated macrophage infiltration. Further loss-of-function studies revealed that silencing PIPKIγ greatly reduced CCL2 expression at both the mRNA and protein levels, leading to weak chemotaxis of cancer cells to macrophages. Mechanistically, PIPKIγ facilitated PI3K-Akt-mTOR signaling pathway activation to increase STAT3 phosphorylation levels, thus triggering CCL2 transcription to enhance tumor-associated macrophage recruitment. These findings identify the PIPKIγ signaling pathway as a new actor in colorectal cancer immunosuppression and a potential therapeutic target for this common cancer.

Protocadherin-8 promotes invasion and metastasis via laminin subunit γ2 in gastric cancer.

Growing evidence suggests that protocadherins (PCDH) play crucial roles in pathogenesis and progression of cancers, including gastric cancer (GC). Protocadherin-8 (PCDH8) was previously reported to be involved in metastasis of GC, but functional studies yielded inconsistent results and the molecular mechanism remained unknown. The present study aimed to explore the clinical relevance, function and molecular mechanism of PCDH8 in GC. Data from the GEPIA and Kaplan-Meier plotter databases showed that high expression of PCDH8 was significantly correlated with poorer prognosis in GC. Ectopic expression of PCDH8 in GC cells promoted invasion and migration in vitro and metastasis in vivo, and knockdown of PCDH8 inhibited invasion and migration in vitro. RNA sequencing followed by gene set enrichment analysis found a remarkable enrichment in the extracellular matrix receptor interaction pathway, with the expression of laminin subunit γ2 (LAMC2) being significantly increased in the PCDH8-overexpressing group. High expression of LAMC2 was significantly correlated to poor prognosis in GC in GEPIA database. Upregulation of LAMC2 following PCDH8 overexpression was further confirmed by immunohistochemistry in liver metastatic lesions of nude mice. To our knowledge, this is the first report of the metastasis-enhancing property and molecular mechanism through upregulation of LAMC2 of PCDH8 in cancer. High expression of PCDH8 could be used as a biomarker for poor prognosis in clinical practice.

CXCR2 promotes breast cancer metastasis and chemoresistance via suppression of AKT1 and activation of COX2.

Metastasis and chemoresistance are two major causes of breast cancer death. We show here that the chemokine receptor CXCR2 was overexpressed in breast cancer cell lines and tissues. CXCR2 promoted anti-apoptosis, anti-senescence, and epithelial-to-mesenchymal transition (EMT) of breast cancer cells, leading to the enhanced metastasis and chemoresistance. Further study suggested that AKT1 and cyclooxygenase-2 (COX2; PTGS2) might mediate the CXCR2 signaling to inversely control the breast cancer metastasis and chemoresistance through the regulation of EMT, apoptosis, and senescence. Analyses of clinical data indicate that the high expression of CXCR2 was correlated with the high expression of COX2 and the low expression of AKT1, P85α, E-cadherin, and ß-catenin in cancer tissues. Poor outcomes were associated with the high expression of CXCR2 or COX2 while favorable survivals were associated with the high expression of P85α, AKT1, or E-cadherin in all cancer patients. Cox multivariate analysis demonstrated that CXCR2, COX2, and AKT1 could be independent predictors for disease free survivals. All these data suggest that CXCR2 promotes breast cancer metastasis and chemoresistance via suppressing AKT1 and activating COX2. Thus, antagonists of the CXCR2 signaling molecules may be used to treat breast cancer patients particularly with high metastasis and chemoresistance.

Plasma microRNA-based signatures to predict 3-year postoperative recurrence risk for stage II and III gastric cancer.

Our aim was to identify plasma microRNA (miRNA)-based signatures to predict 3-year postoperative recurrence risk for patients with stage II and III gastric cancer (GC), so as to provide insights for individualized adjuvant therapy. Plasma miRNA expression was investigated in three phases, involving 407 patients recruited from three centers. ABI miRNA microarray and TaqMan Low Density Array were adopted in the discovery phase to identify potential miRNAs. Quantitative reverse-transcriptase polymerase chain reaction was used to assess the expression of selected miRNAs. Logistic regression models were constructed in the training set (n = 170) and validated in the validation set (n = 169). Receiver operating characteristic analyses, survival analyses and subgroup analyses were further used to assess the accuracy of the models. We identified a 7 miRNA classifier and 7miR + pathological factors index that provided high predictive accuracy of GC recurrence (area under the curve = 0.725 and 0.841 in the training set; and 0.627 and 0.771 in the validation set). High-risk patients defined by the signatures had significantly shorter disease-free survival and overall survival than low-risk patients. The 7 miRNA classifier is an independent prognostic factor, and could add predictive value to traditional prognostic factors. Subgroup analyses revealed the satisfactory performance persisted regardless of stage, and the two models both displayed high accuracy in stage IIA patients. In conclusion, identified microRNA signature may potentially provide some additional benefit for prediction of disease recurrence in patients with stage II and III GC.

Prognostic significance and functional implication of immune activating receptor NKG2D in gastric cancer.

NKG2D, an activating receptor expressed on CD8+ T lymphocytes, serves as a co-stimulation molecule by engagement with its ligands MICA/B and ULBPs to trigger immune activation against tumors. Currently, the biological function and clinical significance of NKG2D in gastric cancer remains unexplored. The study aims to investigate the expression of NKG2D in gastric cancer in association with clinical prognosis and its biological function. Real-time PCR was used to analyze NKG2D expression in paired cancer and adjacent non-malignant tissues in 139 gastric cancer patients between 2007 and 2010 in Shanghai Cancer Center. NKG2D expression showed no association with any clinical characteristic parameters. High NKG2D level was significantly associated with better outcome (P = 0.018 for OS, P = 0.041 for DFS). Using univariate Cox regression model, high NKG2D mRNA resulted in 43% risk reduction in gastric cancer patients (HR = 0.57, CI (0.36-0.91), P = 0.019). High NKG2D level displayed a significant association with longer OS in the multivariate analysis (HR = 0.59, CI (0.363-0.96), P = 0.034), independent of other prognostic factors including Lauren classification, neural infiltration, vascular/lymphatic invasion, TNM stage. Upon co-incubation with cancer cells, NKG2D expression in CD8+ T cells was markedly down-regulated. Functional study suggested that either blocking NKG2D or its ligand ULBP-2 could suppress tumor-killing activity of CD8+ T cells. Our data showed that NKG2D receptor could be an independent favorable prognostic indicator for gastric cancer. Furthermore, decreased NKG2D expression might be the mechanism underlying immune evasion by tumors in gastric cancer.

MicroRNA-421 regulated by HIF-1α promotes metastasis, inhibits apoptosis, and induces cisplatin resistance by targeting E-cadherin and caspase-3 in gastric cancer.

Hypoxia and dysregulation of microRNAs (miRNAs) have been identified as crucial factors in carcinogenesis. However, the potential mechanisms of HIF-1α and miR-421 in gastric cancer have not been well elucidated. In this study, we found that miR-421 was up-regulated by HIF-1α. Overexpression of miR-421 promoted metastasis, inhibited apoptosis, and induced cisplatin resistance in gastric cancer in vivo and in vitro. E-cadherin and caspase-3 were identified as targets of miR-421. Besides, relative mRNA expression of miR-421 was significantly increased in gastric cancer tumor tissues compared with non-tumor tissues in a cohort of gastric cancer specimens (n=107). The expression of miR-421 was higher in advanced gastric cancers compared with localized ones. Moreover, Kaplan-Meier analysis illustrated that those patients with low levels of miR-421 had a significant longer overall survival (p = 0.006) and time to relapse (p = 0.007). Therefore, miR-421 could serve as an important prognostic marker and a potential molecular target for therapy in gastric cancer.

Implication of combined PD-L1/PD-1 blockade with cytokine-induced killer cells as a synergistic immunotherapy for gastrointestinal cancer.

Cytokine-induced killer (CIK) cells represent a realistic approach in cancer immunotherapy with confirmed survival benefits in the context of metastatic solid tumors. However, therapeutic effects are limited to a fraction of patients. In this study, immune-resistance elements and ideal combination therapies were explored. Initially, phenotypic analysis was performed to document CD3, CD56, NKG2D, DNAM-1, PD-L1, PD-1, CTLA-4, TIM-3, 2B4, and LAG-3 on CIK cells. Upon engagement of CIK cells with the tumor cells, expression of PD-1 on CIK cells and PD-L1 on both cells were up-regulated. Over-expression of PD-L1 levels on tumor cells via lentiviral transduction inhibited tumoricidal activity of CIK cells, and neutralizing of PD-L1/PD-1 signaling axis could enhance their tumor-killing effect. Conversely, blockade of NKG2D, a major activating receptor of CIK cells, largely caused dysfunction of CIK cells. Functional study showed an increase of NKG2D levels along with PD-L1/PD-1 blockade in the presence of other immune effector molecule secretion. Additionally, combined therapy of CIK infusion and PD-L1/PD-1 blockade caused a delay of in vivo tumor growth and exhibited a survival advantage over untreated mice. These results provide a preclinical proof-of-concept for simultaneous PD-L1/PD-1 pathways blockade along with CIK infusion as a novel immunotherapy for unresectable cancers.

COX-2 promotes breast cancer cell radioresistance via p38/MAPK-mediated cellular anti-apoptosis and invasiveness.

Radioresistance is one of the major barriers to improve the survival rate of breast cancer patients. Cyclooxygenase 2 (COX-2) is usually overexpressed in highly invasive and metastatic breast cancer, which may indicate an association with breast cancer radioresistance. The function role of COX-2 was investigated by using a radioresistant breast cancer cell line MDA-MB-231/RR10 and its parental cell line MDA-MB-231 cells before or after COX-2 was silenced by a specific small hairpin RNA (shRNA). The cell proliferation, migration, invasion, colony formation, and apoptosis were measured by CCK-8, scratch-wound, transwell, clone formation assay, and flow cytometry. Protein and mRNA expression were analyzed by Western blot and quantitative reverse transcriptase-polymerase chain reaction. COX-2 is upregulated in MDA-MB-231/RR10 cells compared with in MDA-MB-231 cells, and silencing of COX-2 expression by shRNA in MDA-MB-231/RR10 cells decreases the expression of Bcl-2 and Bcl-XL, but increases the proapoptotic protein BAK, leading to the increased apoptosis following treatment with γ-irradiation in comparison with those in control cells. Silencing of COX-2 also increases the expression of ß-catenin and E-cadherin, two anti-invasion proteins, resulting in reduced cell migration and invasion tested by transwell chambers and wound-healing assays. Further study demonstrated that COX-2-induced radioresistance is negatively regulated through the phosphorylation of p38 at Tyr182, and that the phosphorylation of p38 induced by TNF-alpha reduces the expression of Bcl-2, BCL-XL, but increases ß-catenin and E-cadherin, leading to the decreased invasiveness of cells. Our data suggest that COX-2, p38, Bcl-2, Bcl-XL, ß-catenin, and E-cadherin may be considered as potential therapeutic targets against radioresistant breast cancer.

Expression of CXCL14 and its anticancer role in breast cancer.

CXCL14, also known as breast and kidney-expressed chemokine, was initially identified as a chemokine highly expressed in the kidney and breast. The exact function of CXCL14 in human breast cancer is still unclear, although it has been testified to play an anti-tumor role in other tumors, including head and neck squamous cell carcinoma, lung cancer, prostate cancer, and so on. In this study, we tried to demonstrate the relationship between CXCL14 and breast cancer. CXCL14 expressions were detected by reverse transcription-PCR and western blot in 2 normal breast epithelial cell lines and 6 breast cancer cell lines. The effects of CXCL14 on the proliferation and invasion in vitro were tested using the CXCL14-overexpressing cells (MDA-MB-231HM-CXCL14) which were established by stable transfection. We established an orthotropic xenograft tumor model in SCID mice using the MDA-MB-231HM-CXCL14 cells and explored the influence of CXCL14 overexpression on tumor growth and metastasis in vivo. Furthermore, we detected the protein level of CXCL14 in 208 breast cancer patients by immunohistochemistry and discussed the correlation between CXCL14 and the prognosis of breast cancer. CXCL14 mRNA expression is lower in breast cancer cell lines, and MDA-MB-231HM express the lowest levels of CXCL14 mRNA. Overexpression of CXCL14 inhibited cell proliferation and invasion in vitro and attenuated xenograft tumor growth and lung metastasis in vivo. CXCL14 protein level is positively correlated to the overall survival of all patients as well as the patients with lymph node metastasis, and it has a negative correlation with the lymph node metastasis. Our study showed for the first time that CXCL14 is a negative regulator of growth and metastasis in breast cancer. The re-expression or up-regulation of this gene may provide a novel strategy in breast cancer therapy in the future.