Comprehensive Mutation Profiling of Colorectal Cancer Patients With Lung or Liver Metastasis by Targeted Next-Generation Sequencing.

OBJECTIVES: Primary tumor tissue is often analyzed to search for predictive biomarkers and DNA-guided personalized therapies, but there is an incomplete understanding of the discrepancies in the genomic profiles between primary tumors and metastases, such as liver and lung metastases. METHODS: We performed in-depth targeted next-generation sequencing of 520 key cancer-associated genes for 47 matched primary and metastatic tumor samples which were retrospectively collected. RESULTS: A total of 699 mutations were detected in the 47 samples. The coincidence rate of primary tumors and metastases was 51.8% (n = 362), and compared to patients with liver metastases, patients with lung metastases had a significantly greater coincidence rate (P = .021). The number of specific mutations for the primary tumors and liver and lung metastases was 186 (26.6%), 122 (17.5%), and 29 (4.1%), respectively. Analysis of a patient with all three occurrences, including a primary tumor, liver metastasis, and lung metastasis, indicated a possible polyclonal seeding mechanism for liver metastases. Remarkably, multiple samples from patients with primary and metastatic tumors supported a mechanism of synchronous parallel dissemination from primary tumors to metastatic tumors that were not mediated through pre-metastatic tumors. We also found that the PI3K-Akt signaling pathway significantly altered lung metastases compared to matched primary tumors (P = .001). In addition, patients with mutations in CTCF, PIK3CA, or TP53 and LRP1B, AURKA, FGFR1, ATRX, DNMT3B, or GNAS had larger primary tumor sizes and metastases, especially patients with both LRP1B and AURKA mutations. Interestingly, CRC patients with TP53-disruptive mutations were more likely to have liver metastases (P = .016). CONCLUSION: In this study, we demonstrate significant differences in the genomic landscapes of colorectal cancer patients based on the site of metastasis. Notably, we observe a larger genomic variation between primary tumors and liver metastasis compared to primary tumors and lung metastasis. These findings can be used for tailoring treatments based on the specific metastatic site.

Quantitative proteomics profiling reveals the inhibition of trastuzumab antitumor efficacy by phosphorylated RPS6 in gastric carcinoma.

BACKGROUND: The anti-HER2 antibody trastuzumab is a standard treatment for gastric carcinoma with HER2 overexpression, but not all patients benefit from treatment with HER2-targeted therapies due to intrinsic and acquired resistance. Thus, more precise predictors for selecting patients to receive trastuzumab therapy are urgently needed. METHODS: We applied mass spectrometry-based proteomic analysis to 38 HER2-positive gastric tumor biopsies from 19 patients pretreated with trastuzumab (responders n = 10; nonresponders, n = 9) to identify factors that may influence innate sensitivity or resistance to trastuzumab therapy and validated the results in tumor cells and patient samples. RESULTS: Statistical analyses revealed significantly lower phosphorylated ribosomal S6 (p-RPS6) levels in responders than nonresponders, and this downregulation was associated with a durable response and better overall survival after anti-HER2 therapy. High p-RPS6 levels could trigger AKT/mTOR/RPS6 signaling and inhibit trastuzumab antitumor efficacy in nonresponders. We demonstrated that RPS6 phosphorylation inhibitors in combination with trastuzumab effectively suppressed HER2-positive GC cell survival through the inhibition of the AKT/mTOR/RPS6 axis. CONCLUSIONS: Our findings provide for the first time a detailed proteomics profile of current protein alterations in patients before anti-HER2 therapy and present a novel and optimal predictor for the response to trastuzumab treatment. HER2-positive GC patients with low expression of p-RPS6 are more likely to benefit from trastuzumab therapy than those with high expression. However, those with high expression of p-RPS6 may benefit from trastuzumab in combination with RPS6 phosphorylation inhibitors.

PD-1-Positive Tumor-Associated Macrophages Define Poor Clinical Outcomes in Patients With Muscle Invasive Bladder Cancer Through Potential CD68/PD-1 Complex Interactions.

Tumor-associated macrophages (TAMs) regulate tumor immunity. Previous studies have shown that the programmed cell death protein 1 (PD-1)-positive TAMs have an M2 macrophage phenotype. CD68 is a biomarker of TAMs and is considered to be a poor prognostic marker of several malignancies. Our results show that PD-1-positive TAMs can be a negative survival indicator in patients with muscle-invasive bladder cancer (MIBC), and that the mechanistic effects could result due to a combination of PD-1 and CD68 activity. We analyzed 22 immune cell types using data from 402 patients with MIBC from the TCGA database, and found that a high immune score and M2 TAMs were strongly associated with poor clinical outcomes in patients with MIBC. Further, we analyzed resected samples from 120 patients with MIBC and found that individuals with PD-1-positive TAMs showed a reduction in 5-year overall survival and disease-free survival. Additionally, PD-1-positive TAMs showed a significant association with higher programmed death-ligand 1 (PD-L1) expression, the Ki67 index, the pT stage and fewer CD8-positive T cells. Through the co-immunoprecipitation (co-IP) assay of THP-1 derived macrophages, we found that CD68 can bind to PD-1. The binding of CD68 and PD-1 can induce M2 polarization of THP-1 derived macrophages and promote cancer growth. The anti-CD68 treatment combined with peripheral blood mononuclear cells (PBMC) showed obvious synergy effects on inhibiting the proliferation of T24 cells. Together, these results indicate for the first time that CD68/PD-1 may be a novel target for the prognosis of patients with MIBC.

DDX54 Plays a Cancerous Role Through Activating P65 and AKT Signaling Pathway in Colorectal Cancer.

Colorectal cancer (CRC) is one of the most malignant cancers, and its incidence is still steadily increasing. The DDX RNA helicase family members have been found to play a role in various cancers; however, the role of DDX54 in colorectal cancer is still unclear and needed to be defined. Here, we found DDX54 was overexpressed in CRC tissues by the label-free mass spectrum, which was also verified in tissue microarray of colon cancer, as well as the CRC cell lines and TCGA database. High DDX54 level was correlated with tumor stage and distant metastasis, which always indicated a poor prognosis to the CRC patients. DDX54 could promote the proliferation and mobility of CRC cells through increasing the phosphorylation level p65 and AKT leading to the tumorigenesis. Here, we have preliminarily studied the function of DDX54 in CRC, which would improve our understanding of the underlying biology of CRC and provide the new insight that could be translated into novel therapeutic approaches.

High tumor mutation burden in a patient with metastatic gastric cancer sensitive to trastuzumab: a case report.

Patients with HER2-positive gastric cancer (GC) can benefit from the addition of trastuzumab. However, not all patients with HER2-positive GC respond to trastuzumab. Biomarkers affecting its efficacy in patients with advanced gastric cancer (AGC) are largely unknown. Therefore, classifying GC into molecularly distinct subtypes to accurately distinguish between GC patients who would and would not benefit from trastuzumab is worthwhile. Tumor mutation burden (TMB) is a notable feature in GC and whether TMB influences trastuzumab efficacy is still unknown. Herein, we report the case of a 61-year-old man who was diagnosed with metastatic HER2-positive gastric adenocarcinoma that had spread to the liver (T4aN0M1, stage IV). Esophagogastroduodenoscopy revealed a circular ulcer in the posterior wall of the stomach. A computed tomography (CT) scan revealed a 2-cm diameter liver metastasis. Immunohistochemical analysis of the endoscopic biopsy tumor revealed 3+ positive expression for HER2. Whole-exome sequencing (WES) of the tumor tissue revealed 3,736 somatic mutations in 2,423 genes and a very high TMB of 50.3 mutations/Mb. Immunohistochemistry revealed that the patient had mismatch repair-proficient (pMMR) GC. The patient received first-line trastuzumab-containing chemotherapy, and after 2 courses of sequential metronomic trastuzumab-containing chemotherapy, restaging CT showed that the liver metastasis had disappeared. Following resection, the patient had no recurrence and no new tumor metastasis after a follow-up of period nearly 7 years. This study is the first to report that pMMR GC with a high TMB has a favorable response to trastuzumab. The combination of HER2 positivity and a high TMB may be sufficiently predictive of sensitivity to trastuzumab in AGC.

Bioactive PLGA/tricalcium phosphate scaffolds incorporating phytomolecule icaritin developed for calvarial defect repair in rat model.

BACKGROUND/OBJECTIVES: For treatment of large bone defects challenging in orthopaedic clinics, bone graft substitutes are commonly used for the majority of surgeons. It would be proposed in the current study that our bioactive scaffolds could additionally serve as a local delivery system for therapeutic small molecule agents capable of providing support to enhance biological bone repair. METHODS: In this study, composite scaffolds made of poly (lactic-co-glycolic acid) (PLGA) and tricalcium phosphate (TCP) named by P/T was fabricated by a low-temperature rapid prototyping technique. For optimizing the scaffolds, the phytomolecule icaritin (ICT) was incorporated into P/T scaffolds called P/T/ICT. The osteogenic efficacies of the two groups of scaffolds were compared in a successfully established calvarial defect model in rats. Bone regeneration was evaluated by X-ray, micro-computerised tomography (micro-CT), and histology at weeks 4 and/or 8 post-implantation. In vitro induction of osteogenesis and osteoclastogenesis was established for identification of differentiation potentials evoked by icaritin in primary cultured precursor cells. RESULTS: The results of radiographies and decalcified histology demonstrated more area and volume fractions of newly formed bone within bone defect sites implanted with P/T/ICT scaffold than that with P/T scaffold. Undecalcified histological results presented more osteoid and mineralized bone tissues, and also more active bone remodeling in P/T/ICT group than that in P/T group. The results of histological staining in osteoclast-like cells and newly formed vessels indicated favorable biocompatibility, rapid bioresorption and more new vessel growth in P/T/ICT scaffolds in contrast to P/T scaffolds. Based on in vitro induction, the results presented that icaritin could significantly facilitate osteogenic differentiation, while suppressed adipogenic differentiation. Meanwhile, icaritin demonstrated remarkable inhibition of osteoclastogenic differentiation. CONCLUSION: The finding that P/T/ICT composite scaffold can enhance bone regeneration in calvarial bone defects through facilitating effective bone formation and restraining excessive bone resorption. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: The osteogenic bioactivity of icaritin facilitated PLGA/TCP/icartin composite scaffold to exert significant bone regeneration in calvarial defects in rat model. It might form an optimized foundation for potential clinical validation in bone defects application.

Mitochondrial ROS accumulation inhibiting JAK2/STAT3 pathway is a critical modulator of CYT997-induced autophagy and apoptosis in gastric cancer.

BACKGROUND: Gastric cancer (GC) is a common form of malignant cancer in worldwide which has a poor prognosis. Despite recent improvements in the treatment of GC, the prognosis is not yet satisfactory for GC patients. CYT997, a novel microtubule-targeting agent, recently has been identified to be a promising anticancer candidate for the treatment of cancers; however, the effects of CYT997 in GC remain largely unknown. METHODS: Cell proliferation and apoptosis were detected by CCK8 assay and flow cytometry. The mitochondrial ROS were detected by confocal microscope and flow cytometry. Gastric cancer patient-derived xenograft (PDX) model was used to evaluate its antitumor activity of CYT997 in vivo. RESULTS: CYT997 inhibited gastric cancer cell proliferation and induced cell apoptosis and triggered autophagy. CYT997 induced apoptosis through triggering intracellular mitochondrial ROS generation in GC cells. ROS scavengers N-acetylcysteine (NAC) and Mitoquinone (MitoQ) distinctly weakened CYT997-induced cell cycle G2/M arrest and apoptosis in GC cells. Pretreatment with autophagy inhibitor 3-MA promoted the effect of CYT997 on cells apoptosis. Mechanistically, CYT997 performed its function through regulation of Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in GC cells. In addition, CYT997 inhibited growth of gastric cancer patient-derived xenograft (PDX) tumors. CONCLUSIONS: CYT997 induces autophagy and apoptosis in gastric cancer by triggering mitochondrial ROS accumulation to silence JAK2/STAT3 pathway. CYT997 might be a potential antitumor drug candidate to treat GC.

Integrin ß3 promotes cardiomyocyte proliferation and attenuates hypoxia-induced apoptosis via regulating the PTEN/Akt/mTOR and ERK1/2 pathways.

Objective: Integrin ß3 is one of the main integrin heterodimer receptors on the surface of cardiac myocytes. Our previous studies showed that hypoxia induces apoptosis and increases integrin ß3 expression in cardiomyocytes. However, the exact mechanism by which integrin ß3 protects against apoptosis remains unclear. Hence, the present investigation aimed to explore the mechanism of integrin ß3 in cardiomyocyte proliferation and hypoxia-induced cardiomyocyte apoptosis. Methods: Stable cells and in vivo acute and chronic heart failure rat models were generated to reveal the essential role of integrin ß3 in cardiomyocyte proliferation and apoptosis. Western blotting and immunohistochemistry were employed to detect the expression of integrin ß3 in the stable cells and rat cardiac tissue. Flow cytometer was used to investigate the role of integrin ß3 in hypoxia-induced cardiomyocyte apoptosis. Confocal microscopy was used to detect the localization of integrin ß3 and integrin αv in cardiomyocytes. Results: A cobaltous chloride-induced hypoxic microenvironment stimulated cardiomyocyte apoptosis and increased integrin ß3 expression in H9C2 cells, AC16 cells, and cardiac tissue from acute and chronic heart failure rats. The overexpression of integrin ß3 promoted cardiomyocyte proliferation, whereas silencing integrin ß3 expression resulted in decreased cell proliferation in vitro. Furthermore, knocking down integrin ß3 expression using shRNA or the integrin ß3 inhibitor cilengitide exacerbated cobaltous chloride-induced cardiomyocyte apoptosis, whereas overexpression of integrin ß3 weakened cobaltous chloride-induced cardiomyocytes apoptosis. We found that integrin ß3 promoted cardiomyocytes proliferation through the regulation of the PTEN/Akt/mTOR and ERK1/2 signaling pathways. In addition, we found that knockdown of integrin αv or integrin ß1 weakened the effect of integrin ß3 in cardiomyocyte proliferation. Conclusion: Our findings revealed the molecular mechanism of the role of integrin ß3 in cardiomyocyte proliferation and hypoxia-induced cardiomyocyte apoptosis, providing new insights into the mechanisms underlying myocardial protection.

Low serum gastrin associated with ER+ breast cancer development via inactivation of CCKBR/ERK/P65 signaling.

BACKGROUND: Gastrin is an important gastrointestinal hormone produced primarily by G-cells in the antrum of the stomach. It normally regulates gastric acid secretion and is implicated in a number of human disease states, but how its function affects breast cancer (BC) development is not documented. The current study investigated the suppressive effects of gastrin on BC and its underlying mechanisms. METHODS: Serum levels of gastrin were measured by enzyme-linked immunosorbent assay (ELISA) and correlation between gastrin level and development of BC was analyzed by chi-square test. Inhibitory effects of gastrin on BC were investigated by CCK-8 assay and nude mice models. Expressions of CCKBR/ERK/P65 in BC patients were determined through immunohistochemistry (IHC) and Western blot. Survival analysis was performed using the log-rank test. RESULTS: The results indicated that the serum level of gastrin in BC patients was lower compared with normal control. Cellular and molecular experiments indicated that reduction of gastrin is associated with inactivation of cholecystokinin B receptor (CCKBR)/ERK/P65 signaling in BC cells which is corresponding to molecular type of estrogen receptor (ER) positive BC. Furthermore, we found that low expression of gastrin/CCKBR/ERK /P65 was correlated to worse prognosis in BC patients. Gastrin or ERK/P65 activators inhibited ER+ BC through CCKBR-mediated activation of ERK/P65. Moreover, combination treatment with gastrin and tamoxifen more efficiently inhibited ER+ BC than tamoxifen alone. CONCLUSIONS: We concluded that low serum gastrin is related to increased risk of ER+ BC development. The results also established that CCKBR/ERK/P65 signaling function is generally tumor suppressive in ER+ BC, indicating therapies should focus on restoring, not inhibiting, CCKBR/ERK/P65 pathway activity.

Overexpression of dedicator of cytokinesis 2 correlates with good prognosis in colorectal cancer associated with more prominent CD8+ lymphocytes infiltration: a colorectal cancer analysis.

Recently, dedicator of cytokinesis 2 (DOCK2) has been reportedly exhibited high mutation prevalence in the Asian colorectal cancer (CRC) cohort. However, the expression pattern of DOCK2 and its clinical significance in CRC were still unknown. To characterize the role of DOCK2, a tissue microarray (TMA) containing 481 archived paraffin-embedded CRC specimens was performed by immunohistochemistry. Among which, 54 primary CRC tissues showed high expression of DOCK2 protein, while others were negative. Moreover, DOCK2 expression was positively associated with invasion depth (P < .001) and tumor size (P = .016). Significantly, Kaplan-Meier survival analysis revealed that patients with higher DOCK2 expression had a longer overall survival time (P = .017). Furthermore, univariate and multivariate Cox regression analysis confirmed that DOCK2 is an independent prognostic marker in CRC (P = .049,; HR, 0.519; 95% CI, 0.270 to 0.997). In addition, we observed a strong correlation between the infiltration of CD8+ T lymphocytes and DOCK2 expression (P = .0119). Our findings demonstrated that overexpressed DOCK2 might involve in recruiting CD8+ T lymphocytes and serve as a novel prognostic indicator and indicated a potential therapeutic strategy by restoring DOCK2 for CRC.

Prognostic and predictive values of immune infiltrate in patients with head and neck squamous cell carcinoma.

This study sought to determine whether the in situ tumor-infiltrating immune lymphocytes, as a novel companion to the Immunoscore analysis, could be a promising, valuable prognostic and predictive marker in patients with head and neck squamous cell carcinoma (HNSCC). Total (CD3+) and cytotoxic (CD8+) T lymphocytes were assessed using immunohistochemistry in tumor nests and stroma obtained from patient surgical specimens. The "Immunoscore" methodology has been defined to quantify the amount of in situ immune infiltrate (from I0 to I4). Survival curves were measured using the Kaplan-Meier method, and differences in survival and response to therapy between the groups were estimated using the log-rank test. The prognostic value of the Immunoscore was determined using Cox multivariate analysis. The density and location of CD3+ and CD8+ lymphocytes and the associated Immunoscore correlated significantly with differences in disease-free survival (DFS) and overall survival (OS) (all P < .005). Compared with tumor-node-metastasis (TNM) staging, the Immunoscore was found to have an advantage in predicting survival (P = .000). In addition, a high Immunoscore was associated with the tumors of advanced-stage patients who underwent different treatment regimens. The Immunoscore could be a useful prognostic marker. The measurement of CD3+ and CD8+ cell infiltration may be beneficial in HNSCC patients and may help determine which patients may benefit most from definitive chemoradiotherapy.

Gastrin inhibits gastric cancer progression through activating the ERK-P65-miR23a/27a/24 axis.

BACKGROUND: To test the hypothesis that activated extracellular signal-regulated kinase (ERK) regulates P65-miR23a/27a/24 axis in gastric cancer (GC) and the ERK-P65-miR23a/27a/24 axis plays an important role in the development of GC, and to evaluate the role of gastrin in GC progression and ERK-P65-miR23a/27a/24 axis. METHODS: The component levels of the ERK-P65-miR23a/27a/24 axis in four fresh GC tissues, 101 paraffin-embedded GC tissues and four GC cell lines were determined by Western blotting, immunohistochemistry (IHC) or qRT-PCR. The effects of gastrin on GC were first evaluated by measuring gastrin serum levels in 30 healthy and 70 GC patients and performing a correlation analysis between gastrin levels and survival time in 27 GC patients after eight years of follow-up, then evaluated on GC cell lines, GC cell xenograft models, and patient-derived xenografts (PDX) mouse models. The roles of ERK-P65-miR23a/27a/24 axis in GC progression and in the effects of gastrin on GC were examined. RESULTS: ERK- P65-miR23a/27a/24 axis was proved to be present in GC cells. The levels of components of ERK-P65-miR23a/27a/24 axis were decreased in GC tissue samples and PGC cells. The decreased levels of components of ERK-P65-miR23a/27a/24 axis were associated with poor prognosis of GC, and ERK-P65-miR23a/27a/24 axis played a suppressive role in GC progression. Low blood gastrin was correlated with poor prognosis of the GC patients and decreased expression of p-ERK and p-P65 in GC tissues. Gastrin inhibited proliferation of poorly-differentiated GC (PGC) cells through activating the ERK-P65-miR23a/27a/24 axis. Gastrin inhibited GC growth and enhanced the suppression of GC by cisplatin in mice or PGC cell culture models through activating the ERK-P65-miR23a/27a/24 axis or its components. CONCLUSIONS: ERK-P65-miR23a/27a/24 axis is down-regulated, leading to excess GC growth and poor prognosis of GC. Low gastrin promoted excess GC growth and contributed to the poor prognosis of the GC patients by down-regulating ERK-P65-miR23a/27a/24 axis. Gastrin inhibits gastric cancer growth through activating the ERK-P65-miR23a/27a/24 axis.

Development of Whole Slide Imaging on Smartphones and Evaluation With ThinPrep Cytology Test Samples: Follow-Up Study.

BACKGROUND: The smartphone-based whole slide imaging (WSI) system represents a low-cost and effective alternative to automatic scanners for telepathology. In a previous study, the development of one such solution, named scalable whole slide imaging (sWSI), was presented and analyzed. A clinical evaluation of its iOS version with 100 frozen section samples verified the diagnosis-readiness of the produced virtual slides. OBJECTIVE: The first aim of this study was to delve into the quantifying issues encountered in the development of an Android version. It should also provide insights into future high-resolution real-time feedback medical imaging apps on Android and invoke the awareness of smartphone manufacturers for collaboration. The second aim of this study was to further verify the clinical value of sWSI with cytology samples. This type is different from the frozen section samples in that they require finer detail on the cellular level. METHODS: During sWSI development on Android, it was discovered that many models do not support uncompressed camera pixel data with sufficient resolution and full field of view. The proportion of models supporting the optimal format was estimated in a test on 200 mainstream Android models. Other factors, including slower processing speed and camera preview freezing, also led to inferior performance of sWSI on Android compared with the iOS version. The processing speed was mostly determined by the central processing unit frequency in theory, and the relationship was investigated in the 200-model simulation experiment with physical devices. The camera preview freezing was caused by the lag between triggering photo capture and resuming preview. In the clinical evaluation, 100 ThinPrep cytology test samples covering 6 diseases were scanned with sWSI and compared against the ground truth of optical microscopy. RESULTS: Among the tested Android models, only 3.0% (6/200) provided an optimal data format, meeting all criteria of quality and efficiency. The image-processing speed demonstrated a positive relationship with the central processing unit frequency but to a smaller degree than expected and was highly model-dependent. The virtual slides produced by sWSI on Android and iOS of ThinPrep cytology test samples achieved similar high quality. Using optical microscopy as the ground truth, pathologists made a correct diagnosis on 87.5% (175/200) of the cases with sWSI virtual slides. Depending on the sWSI version and the pathologist in charge, the kappa value varied between .70 and .82. All participating pathologists considered the quality of the sWSI virtual slides in the experiment to be adequate for routine usage. CONCLUSIONS: Limited by hardware and operating system support, the performance of sWSI on mainstream Android smartphones did not fully match the iOS version. However, in practice, this difference was not significant, and both were adequate for digitizing most of the sample types for telepathology consultation.

Clinical significance of TM4SF1 as a tumor suppressor gene in gastric cancer.

Transmembrane-4-L-six-family member-1 (TM4SF1), a tumor-associated antigen, is overexpressed in most epithelial cell carcinomas and a potential target for antibody-mediated therapy. However, the role of TM4SF1 in gastric cancer has not been elucidated. The aim of this study was to investigate the clinical significance of TM4SF1 expression in gastric carcinoma (GC) tissues using 152 GC tissue samples and matched adjacent nontumor tissue samples analyzed by immunohistochemistry, and 13 fresh GC tissue samples analyzed by Western blotting. The results showed that TM4SF1 was heterogeneously expressed in normal gastric mucosa, with a high expression rate in fundus mucosa. Higher levels and strong expression rate of TM4SF1 were associated with GC tissues of higher-grade differentiation. TM4SF1 levels were lower in gastric cancer tissues than gastric noncancerous tissues. Expression of TM4SF1 was not correlated with USP10 (P = 0.157), S100A12 (P = 0.479), p53 (P = 0.249), or Ki67 (P = 0.166) in GC. The expression of TM4SF1 was significantly and negatively correlated with depth of invasion (P = 0.031), nodal metastasis (P = 0.042), TNM stage (P = 0.030), and Lauren classification (P = 0.026). There was no significant correlation between TM4SF1 expression and age, gender, tumor size, or distant metastasis (P > 0.05). The expression of TM4SF1 was associated with well overall survival (P = 0.0164). The 5-year survival rate for patients with GC showing TM4SF1 positive was 58.82% (10/17), and the median survival time was 78 months, higher than that (12.90%, 12/93) of patients who were TM4SF1 negative, whose median survival time was 62 months. These data suggested that low expression of TM4SF1 is associated with carcinogenesis and development, tumor progression and invasion of gastric cancer, and poor overall survival of patients with GC. TM4SF1 is a tumor suppressor for GC and a novel prognostic marker for patients with GC.

N-stearoyltyrosine protects primary cortical neurons against oxygen-glucose deprivation-induced apoptosis through inhibiting anandamide inactivation system.

N-stearoylthrosine (NST), a synthesized anandamide (AEA) analogue, plays a neuroprotective role in neurodegenerative diseases and cerebrovascular diseases. Several studies have demonstrated that the endocannabinoids systems (ECS) are involved in the neuroprotective effects against cerebral ischemic injury. Oxygen-glucose deprivation (OGD)-induced neuronal injury elevated the levels of endocannabinoids and activated ECS. This research was conducted to investigate the neuroprotective effect of NST against OGD-induced neuronal injury in cultured primary cortical neurons and the potential mechanism involved. Cortical neurons were treated with NST at indicate concentrations for 30min prior to injury and OGD injured neurons were incubated with normal conditions for 0-24h. The best neuroprotective effect of NST against OGD-induced injury occurred at 10µM. All data indicated that the neuroprotective effect of NST against OGD-induced injury resulted from blocking anandamide membrane transporter (AMT) (IC50=11.74nM) and inhibiting fatty acid amide hydrolase activity (FAAH) (IC50=16.54nM). Our findings demonstrated that NST has an important role in cerebral ischemic injury pathological progression through activating cannabinoid receptors by inhibiting AEA inactivation system. These data suggested a potential role for NST in the therapeutic consideration of cerebral ischemic injury. However, inhibition of AEA inactivation system may provide a neuroprotective effect during cerebral ischemic injury.

Expression of AE1/p16 promoted degradation of AE2 in gastric cancer cells.

BACKGROUND: Human anion exchanger 1 and 2 (AE1 and AE2) mediate the exchange of Cl(-)/HCO3 (-) across the plasma membrane and regulate intracellular pH (pHi). AE1 is specifically expressed on the surface of erythrocytes, while AE2 is widely expressed in most tissues, and is particularly abundant in parietal cells. Previous studies showed that an interaction between AE1 and p16 is a key event in gastric cancer (GC) progression, but the importance of AE2 in GC is unclear. METHODS: The relationship among AE1, AE2 and p16 in GC cells was characterized by molecular and cellular experiments. AE2 expression and pHi were measured after knockdown or forced expression of AE1 or p16 in GC cells. The effect of AE2 on GC growth and the correlation of AE2 expression with differentiation and prognosis of GC were also evaluated. The effect of gastrin on AE2 expression and GC growth was investigated in cellular experiments and mouse xenograft models. RESULTS: p16 binds to both AE1 and AE2 simultaneously. AE1 or p16 silencing elevated AE2 expression on the plasma membrane where it plays a role in pHi regulation and GC suppression. AE2 expression was decreased in GC tissue, and these decreased levels were correlated with poor differentiation and prognosis of GC. The low AE2 protein levels are due to rapid ubiquitin-mediated degradation that was facilitated in the presence of p16. Gastrin inhibited the growth of GC cells at least partially through up-regulation of AE2 expression. CONCLUSION: AE1/p16 expression promoted AE2 degradation in GC cells. Gastrin is a potential candidate drug for targeted therapies for AE1- and p16-positive GC.

PCAF acts as a gastric cancer suppressor through a novel PCAF-p16-CDK4 axis.

Gastric cancer (GC) is a leading cause of cancer-related death worldwide and the pathogenesis of GC remains largely unknown. Here, we demonstrate a novel mechanism by which P300/CBP associating factor (PCAF) acts as a tumor suppressor in GC cells. We showed that both PCAF mRNA and protein were downregulated in GC cells, and that this downregulation correlated with poor survival. Meanwhile, the interaction between human anion exchanger 1 (AE1) and p16 is a key event in GC development. We found that PCAF inhibited GC growth by interacting with AE1 and p16 to promote ubiquitin-mediated degradation of AE1 and p16 upregulation and translocation into the nucleus. Binding of nuclear p16 to CDK4 prevented the CDK4-Cyclin D1 interaction to inhibit GC proliferation. Furthermore, reduced PCAF levels in GC cells were associated with intracellular alkalinization and decreased immunity. Together these results suggest that PCAF acts as a GC suppressor through a novel PCAF-p16-CDK4 axis. The downregulation of PCAF expression in GC cells that follows intracellular alkalinization and decreased immune response, indicates that GC therapies should focus on restoring PCAF levels.

The NF-κB p65/miR-23a-27a-24 cluster is a target for leukemia treatment.

p65 is a transcription factor that is involved in many physiological and pathologic processes. Here we report that p65 strongly binds to the miR-23a-27a-24 cluster promoter to up-regulate its expression. As bone marrow-derived cells differentiate into red blood cells in vitro, p65/miR-23a-27a-24 cluster expression increases sharply and then declines before the appearance of red blood cells, suggesting that this cluster is negatively related to erythroid terminal differentiation. Bioinformatic and molecular biology experiments confirmed that the miR-23a-27a-24 cluster inhibited the expression of the erythroid proteome and contributed to erythroleukemia progression. In addition, high level of the p65/miR-23a-27a-24 cluster was found in APL and AML cell lines and in nucleated peripheral blood cells from leukemia patients. Furthermore, anti-leukemia drugs significantly inhibited the expression of the p65/miR-23a-27a-24 cluster in leukemia cells. Administration of the p65 inhibitor parthenolide significantly improved hematology and myelogram indices while prolonging the life span of erythroleukemia mice. Meanwhile, stable overexpression of these three miRNAs in mouse erythroleukemia cells enhanced cell malignancy. Our findings thus connect a novel regulation pathway of the p65/miR-23a-27a-24 cluster with the erythroid proteome and provide an applicable approach for treating leukemia.

Trastuzumab Inhibits Growth of HER2-Negative Gastric Cancer Cells Through Gastrin-Initialized CCKBR Signaling.

BACKGROUND: Administration of trastuzumab, a fully humanized monoclonal antibody targeted to the human epidermal growth factor receptor 2 (HER2, p185), has improved outcomes for patients with HER2-positive gastric cancer (GC), but some relevant issues remain to be investigated and will emerge with new anti-GC drugs. Gastrin is a major gastrointestinal hormone proven to have an inhibitory effect on GC in vitro and in vivo. AIM: To explore the sympathetic role of trastuzumab and gastrin on inhibition of GC. METHODS: The HER2-positive and HER2-negative GC cell lines were treated with trastuzumab, gastrin, or their combination in vitro and in xenograft model. The synergistical role of trastuzumab and gastrin and related mechanisms were investigated. RESULTS: We found the synergistic inhibitory effects of trastuzumab and gastrin on HER2-negative GC cells through the gastrin/cholecystokinin B receptor (CCKBR) pathway. Trastuzumab upregulated CCKBR protein levels but could not initiate its signal transduction, whereas gastrin increased the levels and activation of CCKBR. Molecular experiments indicated that trastuzumab and gastrin co-treatment synergistically enhanced the stability of CCKBR. Moreover, their combined treatment synergistically arrested GC cells at G0/G1 phase, down-regulated levels of GC-related proteins, including anion exchanger 1 (AE1), cyclin D1, ß-catenin, and cytoplasmic p16, and promoted nuclear translocation of p16. In addition, combination treatment upregulated AE2 levels, which are reduced in GC tissues. The in vivo synergistic anti-GC effect of combined treatment was confirmed in xenograft experiments. CONCLUSIONS: Trastuzumab plus gastrin inhibit growth of Her2-negative GC by targeting cytoplasmic AE1 and p16.