M2-phenotype tumour-associated macrophages upregulate the expression of prognostic predictors MMP14 and INHBA in pancreatic cancer.

Pancreatic cancer is one of the most lethal gastrointestinal tumours, the most common pathological type is pancreatic adenocarcinoma (PAAD). In recent year, immune imbalanced in tumour microenvironment has been shown to play an important role in the evolution of tumours progression, and the efficacy of immunotherapy has been gradually demonstrated in clinical practice. In this study, we propose to construct an immune-related prognostic risk model based on immune-related genes MMP14 and INHBA expression that can assess the prognosis of pancreatic cancer patients and identify potential therapeutic targets for pancreatic cancer, to provide new ideas for the treatment of pancreatic cancer. We also investigate the correlation between macrophage infiltration and MMP14 and INHBA expression. First, the gene expression data of pancreatic cancer and normal pancreatic tissue were obtained from The Cancer Genome Atlas Program (TCGA) and The Genotype-Tissue Expression public database (GTEx). The differentially expressed immune-related genes between pancreatic cancer samples and normal sample were screened by R software. Secondly, univariate Cox regression analysis were used to evaluate the relationship between immune-related genes and the prognosis of pancreatic cancer patients. A polygenic risk score model was constructed by Cox regression analysis. The prognostic nomogram was constructed, and its performance was evaluated comprehensively by internal calibration curve and C-index. Using the risk model, each patient gets a risk score, and was divided into high- or low- risk groups. The proportion of 22 types of immune cells infiltration in pancreatic cancer samples was inferred by CIBERSOFT algorithm, correlation analysis (Pearson method) was used to analyse the correlation between the immune-related genes and immunes cells. Then, we applied macrophage conditioned medium to culture pancreatic cancer cell line PANC1, detected the expression of MMP14 and INHBA by qRT-PCR and Western blot methods. Knock-down MMP14 and INHBA in PANC1 cells by transfected with shRNA lentiviruses. Detection of migration ability of pancreatic cells was done by trans-well cell migration assay. A subcutaneous xenograft tumour model of human pancreatic cancer in nude mice was constructed. In conclusion, an immune-related gene prognostic model was constructed, patients with high-risk scores have poorer survival status, M2-phenotype tumour-associated macrophages (TAMs) up-regulate two immune-related genes, MMP14 and INHBA, which were used to establish the prognostic model. Knock-down of MMP14 and INHBA inhibited invasion of pancreatic cancer.

Tumor-associated macrophages promote the metastasis and growth of non-small-cell lung cancer cells through NF-κB/PP2Ac-positive feedback loop.

Non-small-cell lung cancer (NSCLC), with its aggressive biological behavior, is one of the most diagnosed cancers. Tumor-associated inflammatory cells play important roles in the interaction between chronic inflammation and lung cancer, however the mechanisms involved are far from defined. In the present study, by developing an orthotopic NSCLC mouse model based on chronic inflammation, we proved that an inflammatory microenvironment accelerated the growth of orthotopic xenografts in vivo. Tumor-associated macrophages, the most abundant population of inflammatory cells, were identified. Treatment with macrophage-conditioned medium (MCM) promoted the growth and migration of NSCLC cells. Using bioinformatics analysis, we identified downregulated PP2Ac expression in NSCLC cells upon treatment with MCM. We further confirmed that this downregulation was executed in an NF-κB pathway-dependent manner. As IκB kinase (IKK) has been proved to be a substrate of PP2Ac, inhibition on PP2Ac could result in amplification of NF-κB pathway signaling. Overexpression of PP2Ac, or the dominant-negative forms of IKK or IκB, attenuated the acceleration of growth and metastasis by MCM. Using bioinformatics analysis, we further identified that CXCL1 and COL6A1 could be downstream of NF-κB/PP2Ac pathway. Luciferase assay and ChIP assay further confirmed the location of response elements on the promoter regions of CXCL1 and COL6A1. Elevated CXCL1 facilitated angiogenesis, whereas upregulated COL6A1 promoted proliferation and migration.

The prognostic roles of red blood cell-associated indicators in patients with resectable gastric cancers.

BACKGROUND: Gastric cancer (GC) is one of the leading causes of cancer-related death worldwide. This study was designed to investigate the prognostic values of red blood cell (RBC)-associated indicators, including RBC, hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and RBC distribution width (RDW) in resectable GC patients. METHODS: In this retrospective study, a total of 104 pathologically confirmed GC patients were recruited. These cases were divided into two groups according to the median values of pretreatment RBC, HGB, HCT, MCV, MCH, MCHC, or RDW. To evaluate the changes in RBC-associated indicators values after treatment, we introduced the concept of post-/pre-treatment ratios (≤1 suggested RBC, HGB, HCT, MCV, MCH, MCHC, or RDW values were not increased after therapy, while >1 represented those in increased levels). RESULTS: The lower pretreatment MCHC levels were correlated with worse overall survival (OS), while pretreatment levels of RBC, HGB, HCT, MCV, MCH, or RDW were not. The whole course of treatment (surgery plus adjuvant chemotherapy) significantly decreased the values of MCHC, and increased the values of MCV and RDW, whereas it had no obvious effects on the values of RBC, HGB, HCT, or MCH. Patients with post-/pre-treatment MCV ratio >1 had an increased survival ratio. Meanwhile, post-/pre-treatment RBC, HGB, HCT, MCH, MCHC, or RDW ratios were not correlated with outcomes. Multivariate Cox regression analysis revealed that the American Joint Committee on Cancer (AJCC) stage (III), and lower pretreatment MCHC levels were independent risk factors affecting OS. The receiver operating characteristic (ROC) curve analysis showed that an MCHC value of 341.98 g/L was the optimal cutoff value for prognosis, with a sensitivity of 58.3% and a specificity of 75.0%. CONCLUSIONS: Pretreatment MCHC levels could become a potential prognostic factor for resectable GC.

FOLFIRINOX regulated tumor immune microenvironment to extend the survival of patients with resectable pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly malignant tumors worldwide due to its ineffective diagnosis and poor prognosis. The longest median overall survival (OS) to PDAC patients has been provided by FOLFIRINOX. It is essential to identify the mechanisms of FOLFIRINOX to gain new insights for the treatment of PDAC. METHODS: We compared gene expression levels of PDAC patients who received neoadjuvant FOLFIRINOX prior to surgery with those of patients who received no neoadjuvant chemotherapy. Bioinformatics analysis was applied to screen differentially expressed genes (DEGs). Three microarray data sets were downloaded to analyze gene expression data between PDAC and adjacent non-tumor tissues. Overlapping DEGs were subjected to Kaplan-Meier survival analysis. The genes relating to poor outcomes and would be decreased after FOLFIRINOX were input into the Oncomine, University of Alabama Cancer (UALCAN), and LinkedOmics databases to analyze the gene expression and regulation networks. RESULTS: A total of 83 differentially expressed genes (DEGs) were screened and subjected to bioinformatics analysis, which indicated FOLFIRINOX influenced the immune microenvironment of PDAC. Seventy-three genes significantly associated with the OS of PDAC patients. A Venn diagram revealed CXCL5 and PLAU were related to poor outcomes and would decrease after FOLFIRINOX chemotherapy of PDAC patients. It turned out that CXCL5 participated in the immune response-regulating signaling pathway in PDAC patients. CONCLUSIONS: FOLFIRINOX regulated tumor immunity by reducing expression of the immunosuppressive gene CXCL5, laying a foundation for further study of combination therapy of FOLFIRINOX and immunotherapy.

Inflammation Promotes Progression of Pancreatic Cancer Through WNT/ß-Catenin Pathway-Dependent Manner.

OBJECTIVE: Identify the molecular mechanism of inflammatory stimuli induced pancreatic cancer progression. METHODS: RNA-seq, microarray assay and bioinformatics analyses were used to identify differentially expressed genes. Immunohistochemical staining was performed to evaluate CD68, CD163, ß-catenin, CD103, CCL3 markers. Quantitative real-time polymerase chain reaction (qRT-PCR), luciferase reporter assay, apoptosis assay, wound healing assay and immunofluorescence were performed to study the relationship of inflammatory stimuli and WNT/ß-catenin pathway. RESULTS: Differentially expressed genes of macrophage-conditioned medium-treated pancreatic cancer cells were related with WNT/ß-catenin pathway. Inflammatory stimuli could activate WNT/ß-catenin signaling pathway. In 106 pancreatic cancer patients, nuclear ß-catenin expression of CD68-high group was much higher than CD68-low group (P < 0.05), as same as CD163 (P < 0.05). Inflammatory stimuli downregulated the expression of CCL3 via WNT/ß-catenin pathway and inhibited the chemotaxis of CD103 dendritic cells. Six pancreatic cancer prognosis associating genes were upregulated by inflammatory stimuli via WNT/ß-catenin pathway. Transforming growth factor-ß promoted malignant biological behavior of pancreatic cancer cells through WNT/ß-catenin pathway-dependent mechanism. CONCLUSIONS: Our present study provided a novel mechanism involved in the inflammation-driven cancer progression through tumor immune escape and downstream gene regulation of WNT/ß-catenin pathway-dependent manner.

Identification of key genes and pathways downstream of the ß-catenin-TCF7L1 complex in pancreatic cancer cells using bioinformatics analysis.

As a key component of the Wnt signaling pathway, the ß-catenin-transcription factor 7 like 1 (TCF7L1) complex activates transcription and regulates downstream target genes that serve important roles in the pathology of pancreatic cancer. To identify associated key genes and pathways downstream of the ß-catenin-TCF7L1 complex in pancreatic cancer cells, the current study used the gene expression profiles GSE57728 and GSE90926 downloaded from the Gene Expression Omnibus. GSE57728 is an array containing information regarding ß-catenin knockdown and GSE90926 was developed by high throughput sequencing to provide information regarding TCF7L1 knockdown. Subsequently, differentially expressed genes (DEGs) were sorted separately and the shared 88 DEGs, including 37 upregulated and 51 downregulated genes, were screened. Clustering analysis of these DEGs was performed by heatmap analysis. Functional and pathway enrichment analyses were then performed using FunRich software and Database for Annotation, Visualization and Integrated Discovery, which revealed that the DEGs were predominantly enriched in terms associated with transport, transcription factor activity, and cytokine and chemokine mediated signaling pathway process. A DEG-associated protein-protein interaction (PPI) network, consisting of 58 nodes and 171 edges, was then constructed using Cytoscape software and the 15 genes with top node degrees were selected as the hub genes. Overall survival (OS) analysis of the 88 DEGs was performed and the relevant gene expression datasets were downloaded from The Cancer Genome Atlas. Consequently, three upregulated and seven downregulated genes were identified to be associated with prognosis. Furthermore, high expression levels of five downregulated genes, including CXCL5, CYP27C1, FUBP1, CDK14 and TRIM24, were associated with worse OS. In addition, CDK14 and TRIM24 were revealed as hub genes in the PPI network and both were confirmed to be involved in the Wnt/ß-catenin pathway and phosphoinositide 3-kinase/Akt signaling pathway. Promoter analysis was also applied to the five downregulated DEGs associated with prognosis, which revealed that TCF7L1 may serve as a transcription factor of the DEGs. In conclusion, the genes and pathways identified in the current study may provide potential targets for the diagnosis and treatment of pancreatic cancer.

Prognostic evaluation of resectable colorectal cancer using platelet-associated indicators.

Colorectal cancer (CRC) represents the third most common malignancy worldwide. The aim of the present study was to investigate the predictive values of platelet-associated indicators, including platelet count (PLT), plateletcrit (PCT), mean platelet volume (MPV) and platelet distribution width (PDW) in patients with resectable CRC. The current retrospective study included 153 patients who were pathologically diagnosed with resectable CRC. The patients were divided into two groups according to the median value of PLT, PCT, MPV or PDW. To evaluate the changes in PLT, PCT, MPV and PDW following resection and adjuvant chemotherapy, the concept of post-/pre-treatment PLT, PCT, MPV and PDW ratios was introduced, where <1 indicated decreased PLT, PCT, MPV and PDW values after treatment, and where ≥1 suggested stable or increased values. It was revealed that a low MPV prior to treatment correlated with a higher tumor stage. Surgery significantly decreased MPV, but had no impact on PLT, PCT or PDW. Adjuvant chemotherapy significantly decreased PLT and PCT, increased MPV and had no effect on PDW. After the whole course of treatment (surgery combined with adjuvant chemotherapy), PLT, PCT and PDW were significantly decreased. Kaplan-Meier plots illustrated that patients with a post-/pre-treatment MPV ratio <1 had poorer overall survival (OS), whereas the post-/pre-treatment ratios for PLT, PCT and PDW did not correlate with patient outcome. Multivariate Cox regression analysis revealed that sex, tumor size and the post-/pre-treatment MPV ratio were prognostic factors for OS. Therefore, the present results may suggest MPV as a potential prognostic factor in resectable CRC.

Prognostic Values of Platelet-Associated Indicators in Resectable Lung Cancers.

BACKGROUND: Lung cancer is the leading cause of cancer death. Platelet-related indictors, including platelet count, plateletcrit, mean platelet volume, and platelet distribution width, not only associate with morphology and functions of platelet but also correlate with tumor development and metastasis. In the present study, we investigated the values of platelet-related indictors in the prognosis evaluation of resectable lung cancers. METHODS: In total, 101 patients with resectable lung cancer were recruited in this study. Patients were divided into 2 groups according to the median pretreatment values. To evaluate the individual value changes after treatment, we introduced the concept of post-/pretreatment ratio (≤1 indicated value was not increased after treatment, while >1 suggested increased value). RESULTS: The high pretreatment platelet count level was correlated with larger tumor size. High pretreatment plateletcrit level was associated with more lymph nodes metastasis. Patients with high pretreatment plateletcrit level had worse overall survival, whereas pretreatment platelet count, mean platelet volume, and platelet distribution width levels were not correlated with outcomes. Surgery had no impact on the values of platelet count, plateletcrit, mean platelet volume, or platelet distribution width. Adjuvant chemotherapy significantly decreased the values of platelet count and plateletcrit, whereas it had no effect on the values of mean platelet volume or platelet distribution width. Whole course of treatment (surgery combined with adjuvant chemotherapy) significantly decreased the values of platelet count and platelet distribution width, whereas it had no effect on the values of plateletcrit or mean platelet volume. Post-/pretreatment platelet count, plateletcrit, mean platelet volume, and platelet distribution width ratios were not correlated with outcomes. Univariate analyses demonstrated that American Joint Committee on Cancer stage and pretreatment plateletcrit level were significant risk factors for prognosis. Cox regression analysis revealed that no factor independently associated with worse survival. CONCLUSION: Pretreatment plateletcrit level could be a potential prognostic factor in resectable lung cancers.

VEGFR2 promotes tumorigenesis and metastasis in a pro-angiogenic-independent way in gastric cancer.

BACKGROUND: VEGF/VEGFR2 pathway is the central therapeutic target in anti-angiogenic treatment in multiple cancers. However, little work has been carried out concerning the pro-malignancy functions of VEGFR2 that are independent of its pro-angiogenesis effects in gastric cancer. Here, we demonstrated that VEGFR2 up-regulation in gastric cancer tissues was a prognostic marker for poor disease-free survival and overall survival of gastric cancer patients. METHODS: Immunohistochemistry was used to detect VEGFR2 and VTN expressions in specimens. Kaplan-Meier curves were constructed for survival analysis. Stably knockdown cell lines and overexpression cell lines were constructed by small interfering RNA and plasmids transfection. Real-time PCR and Western blot were used to confirm the expressions of target genes at both RNA and protein levels. Cell proliferation was measured by using Cell Counting Kit-8 and xenograft models. Microarray and bioinformatic analysis were also performed to identify the relationship between Vitronectin (VTN) and VEGFR2. RESULTS: When overexpressed in gastric cancer cells, VEGFR2 increased cellular proliferation and invasion in vitro and tumor formation in xenograft models. By using integrating microarray and bioinformatic analysis, we identifiedVTN as a downstream of VEGFR2 pathway. In gain- and loss-of function analysis in gastric cancer cells, VTN was further verified in consistent with VEGFR2 in expression levels and in regulating cell growth and motility in vitro and in vivo. Moreover, in gastric cancer samples, VTN was as also revealed as a poor prognostic factor. CONCLUSIONS: Our present findings defined a novel activity for VEGFR2 in promoting tumorogenicity, motility and indicating a poor survival in gastric cancer beyond its known pro-angiogenic effects. IMPLICATIONS: Our present findings defined a novel activity for VEGFR2 in promoting tumorogenicity, motility and indicating a poor survival in gastric cancer beyond its known pro-angiogenic effects, which may provide a new and valuable target for design of therapies for intervention and a new cognitive perspective for the anti-angiogenesis therapies.

Prognostic evaluation of patients with resectable lung cancer using systemic inflammatory response parameters.

Lung cancer is one of the leading causes of cancer-associated mortality. C-reactive protein (CRP), albumin (ALB), globulin (GLB), lactate dehydrogenase (LDH), neutrophil-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) have been identified as general parameters for systemic inflammatory response (SIR). Furthermore, these parameters are also associated with tumor development and metastasis. The present study aimed to investigate the predictive values of these SIR parameters in patients with resectable lung cancer. In total, 101 patients with resectable lung cancer were recruited in the present study. The patients were divided into two groups according to the median value of pre-treatment CRP, ALB, GLB, LDH, NLR or PLR values. The post-/pre-treatment ratios were defined as the ratio of pre-treatment blood parameter values and the corresponding values obtained following therapy. A ratio of ≤1.1 indicated that the values were not increased, while a ratio of >1.1 suggested that the values were increased following treatment. Patients with lower pre-treatment ALB levels had poorer overall survival (OS) rates, whereas GLB, LDH, CRP, NLR or PLR levels were not associated with outcomes. Whole course treatment (surgery combined with adjuvant chemotherapy) significantly increased the value of ALB, but decreased the value of NLR, whereas it had no effect on the values of LDH, CRP or PLR. Post-/pre-treatment LDH and PLR were associated with outcomes. Post-/pre-treatment ALB, GLB, CRP and NLR were not associated with outcomes. Multivariate analysis revealed that a low pre-treatment ALB level and increased post-/pre-treatment PLR were independent risk factors affecting OS. The receiver operating characteristic curve analysis demonstrated that an ALB value of 47.850 g/l was considered to be the optimal cut-off value for prognosis; the sensitivity was 28.8% and specificity was 95.9%. It was suggested that the pre-treatment ALB and post-/pre-treatment PLR may be potential prognostic factors in resectable lung cancer.

Values of applying white blood cell counts in the prognostic evaluation of resectable colorectal cancer.

The count and classification of white blood cells (WBCs) may be used as prognostic markers in certain types of cancer. The present study investigated the prognostic potential of the counts of WBCs, including lymphocytes (LYs), monocytes (MOs), neutrophils (NEs), eosinophils (EOs) and basophils (BAs), in the prognosis of resectable colorectal cancer. The present study recruited 153 resectable colorectal cancer cases retrospectively, which were pathologically confirmed. All patients were divided into two groups, according to the median value of LY (low LY, ≤1.632x109/l or high LY, >1.632x109/l), MO (low MO, ≤0.330x109/l or high MO, >0.330x109/l), NE (low NE, ≤3.600x109/l or high NE, >3.600x109/l), EO (low EO, ≤0.085x109/l or high EO, >0.085x109/l), BA (low BA, ≤0.010x109/l or high BA, >0.010x109/l), or WBC (low WBC, ≤5.780x109/l or high WBC, >5.780x109/l). To evaluate the alterations in WBC counts following surgery and adjuvant chemotherapy; all samples received oxiplatin and capecitabine (XELOX) for 6­8 cycles or 5­fluorouracil, leucovorin and oxaliplatin (mFOLFOX6) for 10­12 cycles. XELOX included oxaliplatin administered intravenously at a dose of 130 mg/m2 on day 1 and 850­1,250 mg/m2 capecitabine twice daily for days 1­14, repeated every 3 weeks. mFOLFOX6 included oxaliplatin administered intravenously at a dose of 85 mg/m2, 400 mg/m2 leucovorin and 400 mg/m2 5­FU on day 1 followed by 1,200 mg/m2/days continuous infusion for 2 days (in total, 2,400 mg/m2 over 46­48 h), repeated every 2 weeks. The present study investigated the post/pre­treatment of LY, MO, NE, EO, BA and WBC ratios (≤1 indicated that LY, MO, NE, EO, BA and WBC counts were not increased following therapy; whereas, >1 suggested increased counts). Kaplan­Meier curves were constructed to demonstrate overall survival (OS). A multivariate and univariate logistic regression analyses model was employed to identify the independent risk factors. Low pre­treatment BA counts were associated with larger tumor size (>5 cm); pre­treatment BA levels were positively associated with OS. Surgery significantly decreased the count of BAs and increased the count of EOs; whereas, no effect was observed on LYs, MOs, NEs or WBCs. Adjuvant chemotherapy markedly decreased the counts of LY, NE and WBC; whereas, no notable effects on MOs, EOs or BAs were observed. Whole course treatment (surgery combined with adjuvant chemotherapy) significantly decreased the values of LY, NE and WBC; however, increased the value of EO; no effects on the MO or BA counts were observed. An increased post­/pre­treatment NE ratio suggested poorer prognosis. Multivariate Cox regression analysis revealed that sex, tumor size, pre­treatment BA count and the post­/pre­treatment NE ratio were independent prognostic factors affecting OS. The results of the present study suggested that the pre­treatment BA count and post­/pre­treatment NE ratio may be potential prognostic factors for resectable colorectal cancer.

Prognostic values of platelet-associated indicators in advanced breast cancer.

BACKGROUND: Breast cancer is the leading cause of cancer death in the female population. Platelet-related indicators can be used as prognostic markers of cancers. The present study investigated the potential values of platelet count (PLT), plateletcrit (PCT), mean platelet volume (MPV), platelet distribution width (PDW), and platelet to lymphocyte ratio (PLR) in the prognosis of advanced breast cancer (ABC). METHODS: This retrospective study recruited 94 locally advanced or metastatic breast cancer cases who had histologic and cytologic evidence. The patients were divided into two groups according to the median baseline values of PLT, PCT, MPV, PDW, and PLR. To evaluate the individual value changes after treatment, we introduced the concept of post/pre-treatment ratio (≤1 indicated value was not increased after treatment, while >1 suggested increased value). Responses to chemotherapy, including partial response (PR), stable disease (SD), and progressive disease (PD), were recorded. Kaplan-Meier curves were constructed to show overall survival (OS). Univariate and multivariate Cox regression analysis models were employed to identify the independent risk factors. RESULTS: A lower baseline PCT level was correlated with a better OS. A lower baseline PLT level and a higher baseline PDW level were related to better chemotherapeutic efficacy of breast cancer patients. Univariate analysis and multivariate analysis both revealed that a higher baseline PCT level was an independent prognostic factor for OS. CONCLUSIONS: PCT level may be a potential prognosis factor for ABC.

Down-regulated hsa_circ_0067934 facilitated the progression of gastric cancer by sponging hsa-mir-4705 to downgrade the expression of BMPR1B.

BACKGROUND: Gastric cancer is the third most lethal cancer worldwide. Finding a novel marker is essential to targeted therapy and the diagnosis of gastric cancer. As newly discovered markers, circRNAs have aroused widespread attention on a global scale. Our research aims to understand the role of circRNAs in gastric cancer and to explore the underlying pathogenesis. METHODS: Raw expression data of circRNAs were obtained from the GEO database. Integrated bioinformatics analysis was used to screen differentially expressed circRNAs (DECs) by RobustRankAggreg package. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed to predict the functions of DECs. Then, the miRNAs and mRNAs at the downstream of DECs were predicted. Expression data of miRNAs and mRNAs were downloaded from The Cancer Genome Atlas (TCGA). The aberrantly expressed miRNAs and mRNAs were selected using the edgeR package. RESULTS: Four datasets (GSE78092, GSE83521, GSE89143, and GSE93541) were downloaded from the GEO database. Among them, two DECs (hsa_circ_0007991 and hsa_circ_0067934) were screened. The functional analyses of DECs confirmed that they were cancer-related circRNAs. Furthermore, hsa-mir-4705 (miRNA) and BMPR1B (mRNA) at the downstream of hsa_circ_0067934 were found differentially expressed in gastric cancer by expression data from TCGA database. CONCLUSIONS: Our study discovered the critical roles of hsa_circ_0007991 and hsa_circ_0067934 in the development of gastric cancer, and they could be novel markers for targeted therapy and assist the diagnosis of early-stage gastric cancer. Moreover, we discovered that the hsa_circ_0067934/hsa-mir-4705/BMPR1B axis might be involved in the carcinogenesis of gastric cancer.

The combination of cantharidin and antiangiogenic therapeutics presents additive antitumor effects against pancreatic cancer.

Cantharidin, one of the active components of mylabris, is believed to have antitumor activity. Cantharidin selectively inhibits protein phosphatase 2A (PP2A), which can repress multiple oncogenic kinases (ERK, JNK, PKC, and NF-κB). Researches in vitro have shown that cantharidin suppresses cell viability and metastasis in pancreatic cancer cells. This study aims to investigate the effects of cantharidin on pancreatic cancer xenografts in vivo. Xenograft models were established using cells stably expressing luciferase. Xenograft growth was evaluated by living imaging. Gene expression was determined using a microarray, real-time PCR, a RayBiotech antibody array, and the Milliplex assay. Surprisingly, cantharidin significantly accelerated xenograft growth. Living imaging showed a rapid distribution of D-luciferin in cantharidin-treated xenografts, suggesting a rich blood supply. Immunohistochemistry confirmed increased angiogenesis. Microarray and antibody array identified upregulated proangiogenic and downregulated antiangiogenic factors. The Milliplex assay suggested elevated secretion of IL-6, IL-8, TNF-α, and VEGF. Inhibitors of ERK, JNK, PKC, and NF-κB pathway attenuated the cantharidin-induced changes to proangiogenic gene expression. PKC pathway-inhibiting tamoxifen or antiangiogenic therapeutics, including Ginsenoside Rg3, bevacizumab, Apatinib, and Endostar, antagonized the proangiogenic effect of cantharidin or its derivatives. These regimens presented remarkable additive antitumor effects in vivo. Although cantharidin presents antitumor effects in vitro and has been applied in clinical practice, we revealed an unfavorable proangiogenic side effect. We recommend that the clinical application of cantharidin should be performed on the premise of antivascularization therapy.

The radiotherapy-sensitization effect of cantharidin: Mechanisms involving cell cycle regulation, enhanced DNA damage, and inhibited DNA damage repair.

BACKGROUND: Cantharidin is an inhibitor of protein phosphatase 2 A (PP2A), and has been frequently used in clinical practice. In our previous study, we proved that cantharidin could arrest cell cycle in G2/M phase. Since cells at G2/M phase are sensitive to radiotherapy, in the present study, we investigated the radiotherapy-sesitization effect of cantharidin and the potential mechanisms involved. METHODS: Cell growth was determined by MTT assay. Cell cycle was evaluated by flow cytometry. DNA damage was visualized by phospho-Histone H2A.X staining. Expression of mRNA was tested by microarray assay and real-time PCR. Clinical information and RNA-Seq expression data were derived from The Cancer Genome Atlas (TCGA) pancreatic cancer cohort. Survival analysis was obtained by Kaplan-Meier estimates. RESULTS: Cantharidin strengthened the growth inhibition effect of irradiation. Cantharidin drove pancreatic cancer cells out of quiescent G0/G1 phase and arrested cell cycle in G2/M phase. As a result, cantharidin strengthened DNA damage which was induced by irradiation. Moreover, cantharidin repressed expressions of several genes participating in DNA damage repair, including UBE2T, RPA1, GTF2HH5, LIG1, POLD3, RMI2, XRCC1, PRKDC, FANC1, FAAP100, RAD50, RAD51D, RAD51B and DMC1, through JNK, ERK, PKC, p38 and/or NF-κB pathway dependent manners. Among these genes, worse overall survival for pancreatic cancer patients were associated with high mRNA expressions of POLD3, RMI2, PRKDC, FANC1, RAD50 and RAD51B, all of which could be down-regulated by cantharidin. CONCLUSION: Cantharidin can sensitize pancreatic cancer cells to radiotherapy. Multiple mechanisms, including cell cycle regulation, enhanced DNA damage, and inhibited DNA damage repair, may be involved.

The values of applying classification and counts of white blood cells to the prognostic evaluation of resectable gastric cancers.

BACKGROUND: The classifications and counts of white blood cells (WBCs) have been proved to be able to be used as prognostic markers in cancer cases. The present study investigated the potential values of the classifications and counts of WBC, including lymphocyte (LY), monocyte (MO), neutrophil (NE), eosinophil (EO), and basophil (BA) in the prognosis of resectable gastric cancers (GCs). METHODS: This retrospective study recruited 104 resectable GC cases which were pathologically confirmed. The patients were divided into two groups according to the median pre-treatment values. To evaluate the changes in WBC counts and classification after treatment, we introduced the concept of post/pre-treatment ratios (≤ 1 indicated count was not increased after therapy, while > 1 suggested increased count). RESULTS: Pre-treatment NE and total WBC counts were negatively correlated with overall survival (OS). Surgery significantly decreased the level of NE count, but increased the level of EO, whereas had no effect on the levels of LY, MO, BAor total WBC. Adjuvant chemotherapy significantly decreased the level of BA. Whole course of treatment (surgery combined with adjuvant chemotherapy) had no significant effect on the counts of LY, MO, NE, EO, BA or total WBC. Post/pre-treatment ratios of LY, MO NE, EO, BA and total WBC levels had no effects on OS. Univariate analysis indicated that AJCC stage (III) and higher level of pre-treatment total WBC count were prognostic factors affecting OS. Multivariate Cox regression analysis revealed that AJCC stage (III) and higher level of pre-treatment total WBC count were independent prognostic factors. CONCLUSIONS: Pre-treatment NE count and pre-treatment total WBC count may be potential prognostic factors for the prognostic evaluation of GCs.

Ginsenoside Rg3 targets cancer stem cells and tumor angiogenesis to inhibit colorectal cancer progression in vivo.

Anti-angiogenic therapy has been successfully applied to treat colorectal cancer (CRC). Ginsenoside Rg3, derived from the Chinese herb ginseng, has anti-vascularization effects and can inhibit tumor growth and metastasis, and can sensitize cancer cells to chemotherapy. Therefore, in the present study, we investigated whether Rg3 could be appropriate for CRC treatment. Growth of CRC cells was assessed by an MTT (methyl thiazolyl tetrazolium) assay in vitro and using orthotopic xenograft models in vivo. mRNA expression was evaluated using real-time PCR. Protein levels were tested by western blotting, flow cytometry and immunohistochemistry. Migration was determined using a wound-healing assay. Stemness was further confirmed using a plate clone formation assay. We found that Rg3 repressed the growth and stemness of CRC cells both in vitro and in vivo. Rg3 also impaired the migration of CRC cells in vitro. Rg3 downregulated the expressions of angiogenesis-related genes, and repressed the vascularization of CRC xenografts. In addition, Rg3 strengthened the cytotoxicity of 5-Fluorouracil and oxaliplatin against orthotopic xenografts in vivo. Moreover, Rg3 downregulated the expressions of B7-H1 and B7-H3, high expressions of which were associated with reduced overall survival (OS) of CRC patients. Hence, Rg3 not only repressed the growth and stemness of CRC cells, but could also remodel the tumor microenvironment through repressing angiogenesis and promoting antitumor immunity. Therefore, Rg3 could be a novel therapeutic for the CRC treatment.

Genomic characteristics of pancreatic squamous cell carcinoma, an investigation by using high throughput sequencing after in-solution hybrid capture.

Squamous cell carcinoma (SCC) of pancreas is a rare histotype of pancreatic ductal carcinoma which is distinct from pancreatic adenocarcinoma (AC). Although there are standard treatments for pancreatic AC, no precise therapies exist for pancreatic SCC. Here, we screened 1033 cases of pancreatic cancer and identified 2 cases of pure SCC, which were pathologically diagnosed on the basis of finding definite intercellular bridges and/or focal keratin peal formation in the tumor cells. Immunohistochemistry assay confirmed the positive expression of CK5/6 and p63 in pancreatic SCC. To verify the genomic characteristics of pancreatic SCC, we employed in-solution hybrid capture targeting 137 cancer-related genes accompanied by high throughput sequencing (HTS) to compare the different genetic variants in SCC and AC of pancreas. We compared the genetic alterations of known biomarkers of pancreatic adenocarcinoma in different pancreatic cancer tissues, and identified nine mutated genes in SCC of pancreas: C7orf70, DNHD1, KPRP, MDM4, MUC6, OR51Q1, PTPRD, TCF4, TET2, and nine genes (ABCB1, CSF1R, CYP2C18, FBXW7, ITPA, KIAA0748, SOD2, SULT1A2, ZNF142) that are mutated in pancreatic AC. This study may have taken one step forward on the discovery of potential biomarkers for the targeted treatment of SCC of the pancreas.

TNF-α sensitizes chemotherapy and radiotherapy against breast cancer cells.

PURPOSE: Despite new developments in cancer therapy, chemotherapy and radiotherapy remain the cornerstone of breast cancer treatment. Therefore, finding ways to reduce the toxicity and increase sensitivity is particularly important. Tumor necrosis factor alpha (TNF-α) exerts multiple functions in cell proliferation, differentiation and apoptosis. In the present study, we investigated whether TNF-α could enhance the effect of chemotherapy and radiotherapy against breast cancer cells. METHODS: Cell growth was determined by MTT assay in vitro, and by using nude mouse tumor xenograft model in vivo. Cell cycle and apoptosis/necrosis were evaluated by flow cytometry. DNA damage was visualized by phospho-Histone H2A.X staining. mRNA expression was assessed by using real-time PCR. Protein expression was tested by Western blot assay. RESULTS: TNF-α strengthened the cytotoxicity of docetaxel, 5-FU and cisplatin against breast cancer cells both in vitro and in vivo. TNF-α activated NF-κB pathway and dependently up-regulated expressions of CyclinD1, CyclinD2, CyclinE, CDK2, CDK4 and CDK6, the key regulators participating in G1→S phase transition. As a result, TNF-α drove cells out of quiescent G0/G1 phase, entering vulnerable proliferating phases. Treatment of TNF-α brought more DNA damage after Cs137-irradiation and strengthened G2/M and S phase cell cycle arrest induced by docetaxel and cisplatin respectively. Moreover, the up-regulation of RIP3 (a necroptosis marker) by 5-FU, and the activation of RIP3 by TNF-α, synergistically triggered necroptosis (programmed necrosis). Knockdown of RIP3 attenuated the synergetic effect of TNF-α and 5-FU. CONCLUSION: TNF-α presented radiotherapy- and chemotherapy-sensitizing effects against breast cancer cells.

Participation of autophagy in the cytotoxicity against breast cancer cells by cisplatin.

Breast cancer is one of the most common cancers affecting women worldwide. Conventional chemotherapy is still one of the major approaches to the treatment of breast cancer. Autophagy, also termed as type II programmed cell death (PCD), exhibits either a protumorigenic or antitumorigenic function. In the present study, we investigated whether autophagy could be involved in the effect of chemotherapy against breast cancer. Epirubicin, docetaxel, methotrexate, cyclophosphamide, fluorouracil (5-FU) and cisplatin were applied in the present investigation. All of these chemotherapeutics presented cytotoxicity against breast cancer cells. DsRed-LC3 reporter assay revealed that only docetaxel and cisplatin induced autophagy. Autophagy inhibitor 3-methyladenine (3-MA) strengthened the cytotoxicity of docetaxel, yet impaired the cytotoxicity of cisplatin, suggesting that docetaxel stimulates protumorigenic autophagy, while cisplatin-induced autophagy could be antitumorigenic. Real-time PCR revealed that cisplatin upregulated multiple autophagy-related genes, including AMBRA1, ATG3, ATG4C, ATG4D, ATG5, ATG7, ATG13, ATG14, ATG16L2, Beclin1, DRAM1, GABARAP, GABARAPL1, GABARAPL2, HDAC6, IRGM, MAP1LC3B and ULK1, indicating that cisplatin induced autophagy through a multiple mechanism involved manner.