IGFBP7 remodels the tumor microenvironment of esophageal squamous cell carcinoma by activating the TGFβ1/SMAD signaling pathway

Esophageal squamous cell carcinoma (ESCC) is the most common type of esophageal cancer, and its development, growth, and invasiveness are regulated by the tumor microenvironment (TME). Insulin-like growth factor-binding protein-7 (IGFBP7), which is closely related to various tumors, transforming growth factor-β1 (TGFβ1), which is a key signal mediator in oncogenesis, α-smooth muscle actin (α-SMA), and collagen I are important components of the TME. IGFBP7 can upregulate the expression of TGFβ1 and activate the TGFβ1/SMAD signaling pathway, which leads to an increase in collagen I in hepatic stellate cells (HSCs). However, the contribution of IGFBP7 to TGFβ1 and the TME in the progression of ESCC remains unknown. In the present study, we investigated IGFBP7 expression and its effects on TGFβ1 and the TME in ESCC. A total of 45 patients were divided into three groups: early-tumor group (n=15), advanced-tumor group (n=15), and paracancer control group (n=15). The EC109 cell line was cultured and treated with AdIGFBP7 and LvshTGFβ1, and the expression levels of IGFBP7, TGFβ1, α-SMA, collagen I, and p-SMAD2/3 were determined by immunohistochemical staining and western blotting analysis. IGFBP7, TGFβ1, α-SMA, and collagen I were upregulated in the ESCC samples compared with the control samples (P<0.05), and the values peaked in the advanced-tumor group (P<0.05). Compared with the control group, the TGFβ1, α-SMA, p-SMAD2/3, and collagen I proteins were gradually increased from 24 to 72 h in the EC109 cells treated with AdIGFBP7 (P<0.05). Inhibition of TGFβ1 expression in the EC109 cells treated with AdIGFBP7 gradually reduced the expression of α-SMA, collagen I, and p-SMAD2/3 from 24 to 72 h (P<0.05). These findings suggest that increased IGFBP7 may accelerate the progression of ESCC by upregulating TGFβ1, α-SMA, and collagen I via activating the TGFβ1/SMAD signaling pathway, which could remodel the TME.     

The hsa_circ_0007396-miR-767-3p-CHD4 axis is involved in the progression and carcinogenesis of gastric cancer

BackgroundCircular RNAs (circRNAs) have been linked to numerous human cancers, including gastric cancer (GC), in numerous recent investigations. The expression of circRNA and the mechanisms involved in GC are still unknown.MethodsIn this work, Gene Expression Omnibus 2R (GEO2R) online tool was first used to screen 6 candidates of differentially expressed circRNAs in 2 datasets, {"type":"entrez-geo","attrs":{"text":"GSE83521","term_id":"83521"}}GSE83521 and {"type":"entrez-geo","attrs":{"text":"GSE89143","term_id":"89143"}}GSE89143. Then, using Cancer-Specific CircRNA Database (CSCD), the structural loop diagrams of these circRNAs were generated. After combining the Circular RNA Interactome (CRI) and CSCD databases for miRNA co-prediction, a candidate circRNA-miRNA sub-network was successfully created. The expression of these miRNAs was further examined using Cytoscape software, and 2 miRNAs, miR-767-5p and miR-767-3p.ResultsWe used GEO2R to analyze the differential expression of {"type":"entrez-geo","attrs":{"text":"GSE83521","term_id":"83521"}}GSE83521 and {"type":"entrez-geo","attrs":{"text":"GSE89143","term_id":"89143"}}GSE89143 datasets in GEO database. Through the construction of the structural ring diagram of CSCD database, we found that hsa_circRNA_100571, hsa_circRNA_103102, hsa_circRNA_100754, hsa_circRNA_100737, hsa_circRNA_100269, hsa_circRNA_102476, hsa_circRNA_101287 is the final candidate circRNA in GC. MiR-767-5p and miR-767-3p were found to be important miRNAs in GC. The miRNet database indicated their downstream target genes. In various studies, namely central gene screening, correlation analysis, and protein-protein interaction (PPI), we detected chromodomain helicase DNA binding protein 4 (CHD4) as a key potential candidate of hsa-mir-767-3p. Next, we conducted validation of clinical data. We included the clinical data of 100 patients with GC, and found that patients with low CHD4 expression had significantly higher OS and PFS than those with high CHD4 expression (P<0.001, P=0.005). Cox regression analysis showed that low CHD4 expression was an independent risk factor for tumor progression (P=0.001). At the same time, tumor differentiation and chemotherapy also had a certain impact on the progression of GC (all P<0.05). Therefore, CHD4 may provide a promising therapeutic target for the future treatment of GC.ConclusionsWe identified an important hsa_circ_0007396-miR-767-3p-CHD4 axis, which is associated with GC proliferation and carcinogenesis, and may represent a promising therapeutic target for the future cure of GC.     

Comprehensive analysis of immune related lncRNAs in the tumor microenvironment of stage II–III colorectal cancer

BackgroundLong non-coding RNAs (lncRNAs) associated with immunological function have increasingly been found to act as effective prognostic biomarkers of the overall survival (OS) of colorectal cancer (CRC) patients. We sought to identify a signature of immune-related lncRNAs that offered value as a tool for the prospective prognostic evaluation of patients with stage II–III CRC.MethodsThe clinical and gene expression data of CRC patients in The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases was obtained and separated into a training cohort composed of 202 samples, a test cohort of 124 samples from the {"type":"entrez-geo","attrs":{"text":"GSE72970","term_id":"72970"}}GSE72970 dataset, and a validation cohort of 91 samples from the {"type":"entrez-geo","attrs":{"text":"GSE143985","term_id":"143985"}}GSE143985 dataset.ResultsWe firstly evaluated intratumoral immune cell infiltration by conducting a Single-sample gene set enrichment analyses (ssGSEA) analysis to separate patient tumors into those with low immune cell infiltration and those with high immune cell infiltration. We then compared lncRNA and mRNA expression profiles between these two tumor types, leading us to focus on eight lncRNAs identified within the resultant mRNA-lncRNA co-expression network. Multivariate Cox regression models were then utilized to detect an immune-associated lncRNA signature that offered value for prognostic model construction. Functional analyses revealed this lncRNA signature to be associated with key immunological pathways including the JAK-STAT signaling, T cell receptor signaling, and Rap1 signaling pathways.ConclusionsTogether, our results suggest that our immune-related 4 lncRNA signature can reliably predict stage II–III CRC patient prognosis, thereby guiding efforts to better understand this disease and to effectively treat it.     

Identification of a 3-gene signature for predicting the prognosis of stage II colon cancer based on microsatellite status

BackgroundThough colon cancer (CC) is one of the most malignant tumors across the world, CC patients with microsatellite instability-high (MSI-H) in stage II seem to have a better prognosis. However, the molecular mechanisms underlying the phenomena haven’t been elucidated yet.MethodsThis study enrolled 322 CCs with known microsatellite status from {"type":"entrez-geo","attrs":{"text":"GSE143985","term_id":"143985"}}GSE143985, {"type":"entrez-geo","attrs":{"text":"GSE39582","term_id":"39582"}}GSE39582 and {"type":"entrez-geo","attrs":{"text":"GSE92921","term_id":"92921"}}GSE92921 in the Gene Expression Omnibus (GEO) database. Robust rank aggregation (RRA) analysis, univariate Cox regression analysis and multivariate Cox stepwise regression analysis were performed to identify genes and construct risk score signature. Kaplan-Meier and receiver operating characteristic (ROC) curves analyses were used to evaluate the prognostic value of the signature. The potential mechanisms underlying this signature were assessed in the Metascape database, gene set enrichment analysis (GSEA) and immune infiltration analysis.ResultsRRA analysis identified 40 differently expressed genes (DEGs). A 3-gene risk score signature (MKQ signature) associated with disease-free survival (DFS) was generated. DFS was significantly longer in CC patients with lower than higher scores (P=0.0046). The areas under curves (AUCs) of the time-dependent ROC curves of MKQ signature at 1-, 3- and 5-year DFS were 1, 0.963 and 0.961 respectively. Recurrence-free survival (RFS) was significantly longer in patients in {"type":"entrez-geo","attrs":{"text":"GSE39582","term_id":"39582"}}GSE39582 with lower than higher risk scores (P=0.032). The AUCs for 1-, 3- and 5-year RFS in {"type":"entrez-geo","attrs":{"text":"GSE39582","term_id":"39582"}}GSE39582 were 0.63, 0.618 and 0.583, respectively, validating the value of the MKQ signature. Functional annotation and GSEA revealed that the MKQ signature was associated with multiple immune-related pathways. Immune cell infiltration was found to differ in patients differing in the MKQ signature.ConclusionsGene expression and microsatellite status identified a 3-gene signature (MKQ signature) that could facilitate risk-stratified management in patients with stage II CC. Dysregulation of MSMB, KRT23, and QPRT can serve as prognostic markers in stage II CC.     

CENPF as an independent prognostic and metastasis biomarker corresponding to CD4+ memory T cells in cutaneous melanoma

Owing to recent advances in immunotherapies, the overall survival of patients with skin cutaneous melanoma (SKCM) has increased; however, the 5‐year survival rate of metastatic patients remains poor. Skin cutaneous melanoma—upregulated genes were screened via analysis of differentially expressed genes ({"type":"entrez-geo","attrs":{"text":"GSE3189","term_id":"3189"}}GSE3189 and {"type":"entrez-geo","attrs":{"text":"GSE46517","term_id":"46517"}}GSE46517), and metastasis‐related oncogenes were identified via weighted gene coexpression network analysis of the {"type":"entrez-geo","attrs":{"text":"GSE46517","term_id":"46517"}}GSE46517 dataset. As confirmed by the Tumor Immune Estimation Resource, we found highly expressed centromere protein F (CENPF) in SKCM and its metastases. Immunostaining of human melanoma tissues demonstrated high CENPF expression. According to the Kaplan‐Meier survival curve log‐rank test, receiver‐operating characteristic curve, and univariate and multivariate analyses, the Cancer Genome Atlas (TCGA) database suggested CENPF be a typical independent predictor of SKCM. The CIBERSORT algorithm classified the types of the immune cells from {"type":"entrez-geo","attrs":{"text":"GSE46517","term_id":"46517"}}GSE46517 and showed higher proportion of CD4+ memory‐activated T cells in metastatic melanoma. Single‐sample gene set enrichment analysis of TCGA data confirmed the correlation between CENPF and activated CD4+ T cells. Centromere protein F was positively correlated with tumor mutational burden and CD4+ memory T cell markers (interleukin [IL]‐23A, CD28, and CD62L), negatively associated with memory T cell maintenance factors (IL‐7 and IL‐15) by correlation analysis. Moreover, immunofluorescence showed high coexpression of CENPF and IL23A, CD4 in melanoma. Upregulated CENPF might lead to premature depletion of CD4+ memory T cells and immunosuppression. Nomogram indicated CENPF clinical predictive value for 1‐, 3‐, 5‐, and 7‐year melanoma overall survival. Therefore, CENPF plays a vital role in the progression and metastasis of melanoma and can be an effective therapeutic target.     

Intratumoral density of regulatory T cells is a predictor of host immune response and chemotherapy response in colorectal cancer

Regulatory T cells (Tregs) are a subset of CD4+ T lymphocytes known to dampen the host immune response against cancer cells. Within the tumor microenvironment, Tregs are potent facilitators of immune tolerance, and a higher proportion of Tregs compared to cytotoxic T cells predicts a worse outcome in most solid tumors. We studied the association between Treg density, and cancer biology and clinical outcome in colorectal cancer (CRC). We used xCell to estimate intratumoral Tregs in total of 898 CRC patients in the Cancer Genome Atlas (TCGA) and {"type":"entrez-protein","attrs":{"text":"GCE39582","term_id":"1541319275"}}GCE39582 cohorts. High-Treg CRCs enriched immune response-related gene sets; inflammatory response, IFN-γ and IFN-α response, IL2/IL6 signaling, and allograft rejection, and had significantly high infiltration of CD8, CD4, M1 and M2 macrophage, and dendritic cells in both cohorts. While high-Treg CRCs enriched multiple pro-cancer signaling pathways compared to low-Treg CRCs, such as Epithelial Mesenchymal Transition, K-ras, Hypoxia, TGF-β, TNF-α, and angiogenesis, Treg infiltration was surprisingly associated with earlier CRC stage in TCGA. Notably, in two separate cohorts a higher proportion of Tregs predicted an improved response to chemotherapy. In the {"type":"entrez-geo","attrs":{"text":"GSE28702","term_id":"28702"}}GSE28702 cohort, metastatic CRCs with more Tregs showed a significantly better response to mFOLFOX6 versus low-Treg CRC metastases (88.9% response vs. 16.7%, P<0.001). In the {"type":"entrez-geo","attrs":{"text":"GSE72970","term_id":"72970"}}GSE72970 cohort, high-Treg CRCs were found to have a 68.8% response to FOLFOX/FOLFIRI without bevacizumab, compared to 44% response in the low-Treg CRCs. Additionally, high-Treg CRCs were associated with increased expression of immune checkpoint molecules PD-L1/PD-L2, CTLA4, TIGIT and BTLA, implying susceptibility to immunotherapy. We also found that CRCs with higher proportions of Tregs were associated with lower amounts of three microorganisms in the tumor: Lachnoclostridium, flavivirus, and Ornithobacterium. In conclusion, we show that amount of Treg in the tumor is a predictor of host immune response and chemotherapy response in CRC.     

Higher intra-tumoral expression of pro-coagulation genes is a predictor of angiogenesis,epithelial mesenchymal transition and worse patient survival in gastric cancer

Coagulation regulates angiogenesis in cancer, and is associated with tumor development and metastasis. To date, there have been no studies quantifying the state of intra-tumoral coagulation. We measured intra-tumoral coagulation gene expression using the “Hallmark-COAGULATION” gene set in the MSigDB, performing gene set variation analysis and then assigning a “coagulation score” to quantify gene expression. Clinical, histologic, and genetic data were analyzed in 807 gastric cancer patients from the TCGA_STAD and {"type":"entrez-geo","attrs":{"text":"GSE84437","term_id":"84437"}}GSE84437 databases. Tumors with increased expression of pro-coagulation genes were consistently associated with higher AJCC T-categories (p = 0.018), lymph node metastasis (p = 0.036), and stage (p = 0.006) in both cohorts. Patients with high coagulation scores were found to have worse disease-specific survival and overall survival (OS) (p = 0.019 and 0.011, respectively) in TCGA, and worse OS in {"type":"entrez-geo","attrs":{"text":"GSE84437","term_id":"84437"}}GSE84437 cohort (p = 0.012). Higher expression of pro-coagulation genes correlated with increased intra-tumoral angiogenesis, as well as increased proportions of lymphatic and microvascular endothelial cells, endothelial cells, and pericytes, calculated by xCell algorithm. High coagulation scores were significantly associated with low tumor mutation burden, but not with intratumor heterogeneity and homologous recombination deficiency. Gastric cancers with high coagulation scores contained higher amounts of M1 macrophages and dendritic cells, and low numbers of Th1 cells (all P<0.001). Genes for epithelial mesenchymal transition (EMT), myogenesis, apical junction, transforming growth factor (TGF)-β signaling, and angiogenesis were enriched in high coagulation score-gastric cancers (all false discovery rate <0.25). In conclusion, gastric cancers expressing higher levels of pro-coagulation genes demonstrate increased angiogenesis, EMT, TGF-β signaling and worse patient prognosis.     

Analyzing large scale gene expression data in colorectal cancer reveals important clues; CLCA1 and SELENBP1 downregulated in CRC not in normal and not in adenoma

Early detection of colorectal cancer (CRC) increases the chances of survival and reduces the therapeutic problems and costs of treatment. Since molecular biomarkers can help us diagnose colorectal cancer early, we need to identify novel gene for predicting the early stages of tumorigenesis. Here, we integrated five independent CRC gene expression datasets derived from expression profiling by array comparing CRC with normal samples in: {"type":"entrez-geo","attrs":{"text":"GSE21510","term_id":"21510"}}GSE21510, {"type":"entrez-geo","attrs":{"text":"GSE4107","term_id":"4107"}}GSE4107, {"type":"entrez-geo","attrs":{"text":"GSE25071","term_id":"25071"}}GSE25071, {"type":"entrez-geo","attrs":{"text":"GSE15781","term_id":"15781"}}GSE15781 dataset, and {"type":"entrez-geo","attrs":{"text":"GSE8671","term_id":"8671"}}GSE8671 dataset, including 64 samples from 32 patients comparing 32 colonic normal mucosa with 32 colorectal adenoma. To detect genes that expressed differentially in experimental circumstances of these datasets, we used web tool of GEO2R to compare groups of samples in the GEO data series. Furthermore, we constructed the protein-protein interactions network by STRING database for mostly downregulated genes and the expression of their members in PPI network were studied into five datasets separately. Also, the level of expression of selected biomarker genes in different stages of CRC compared to normal was studied. Our data revealed 17 common downregulated genes (average fold change (FC) in five tests ≥6) in CRC in comparison with normal (Test 1 to Test 4) and in adenoma compared with normal (Test 5). Studying of gene expression of PPI network members of these downregulated genes led to identifying of CLCA1, SELENBP1, CWC25, ACOT11, GUCY2C and ALDH1A1 as suppressor genes and PTGS2, PROCR, MOCS3 and NFS1 as oncogenes which respectively downregulated and upregulated in CRC. Since decreasing of gene expression was seen in CRC comparing with normal and due to no different expression seen for these 10 genes in adenoma, they, especially CLCA1 and SELENBP1, could be considered as biomarkers for early detection of CRC. Before using these signature genes in the clinic; however, further validations are required.     

Identification of potential core genes in gastric cancer using bioinformatics analysis

BackgroundGastric cancer is the third leading cause of cancer-related mortality in China. Most patients with gastric cancer have no obvious early symptoms; thus, many of them are in the middle and late stages of gastric cancer at first diagnosis and miss the best treatment opportunity. Molecular targeted therapy is particularly important in changing this status quo.MethodsThree microarray datasets ({"type":"entrez-geo","attrs":{"text":"GSE29272","term_id":"29272"}}GSE29272, {"type":"entrez-geo","attrs":{"text":"GSE33651","term_id":"33651"}}GSE33651, and {"type":"entrez-geo","attrs":{"text":"GSE54129","term_id":"54129"}}GSE54129) were selected from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were screened using GEO2R. The Database for Annotation, Visualization and Integrated Discovery (DAVID) was used to analyze the functional features of these DEGs and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. The protein-protein interaction (PPI) of these DEGs was visualized by Cytoscape software. The expressions of hub genes were evaluated based on Gene Expression Profiling Interactive Analysis (GEPIA). Moreover, we used the online Kaplan-Meier plotter survival analysis tool to evaluate the prognostic values of hub genes. The Target Scan database was used to predict microRNAs that could regulate the target gene, collagen type IV alpha 1 chain (COL4A1). The OncomiR database was used to analyze the expression levels of three microRNAs, as well as the relationships with tumor stage, grade, and prognosis.ResultsWe identified 78 DEGs, including 53 upregulated genes and 25 downregulated genes. The DEGs were mainly enriched in extracellular matrix organization, extracellular structure organization, and response to wounding. Moreover, three KEGG pathways were markedly enriched, including focal adhesion, complement and coagulation cascades, and extracellular matrix (ECM)-receptor interaction. Among these 78 genes, we selected 10 hub genes. The overexpression levels of these hub genes were closely related to poor prognosis and the development of gastric cancer (except for COL3A1, LOX, and CXCL8). Moreover, we found that microRNA-29a-3p, miR-29b-3p, and miR-29c-3p were the potential microRNAs that could regulate the target gene, COL4A1.ConclusionsOur results showed that FN1, COL1A1, TIMP1, COL1A2, SPARC, COL4A1, and SERPINE1 could contribute to the development of novel molecular targets and biomarker-driven treatments for gastric cancer.     

The Down-Regulation of Circ_0059707 in Acute Myeloid Leukemia Promotes Cell Growth and Inhibits Apoptosis by Regulating miR-1287-5p

Acute myeloid leukemia (AML) is the most common type of hematological malignancy. Recently, an increasing number of reports have shown that many circular RNAs can act as effective targets for AML. However, the roles of circ_0059707 in AML remain largely unclear. In this study, we found that the expression levels of circ_0059707 were significantly decreased in AML patients with respect to normal controls (p < 0.001). Low expression levels of circ_0059707 were also associated with a poor prognosis. Furthermore, circ_0059707 overexpression inhibited cell growth and promoted apoptosis in leukemia cells, compared with control cells. Circ_0059707- and empty plasmid-transfected cells were injected subcutaneously into BALB/c nude mice. We found that the tumor volume was significantly lower in mice in the circ_0059707 group than in control mice (p < 0.01). Nuclear pyknosis, nuclear fragmentation, nuclear dissolution, and cell necrosis were observed in the circ_0059707 group by HE staining. CircInteractome analysis showed that 25 microRNAs (miRNAs), including miR-1287-5p, ©-miR-1825, a©hsa-miR-326, may be potential targets for circ_0059707. The expression of these miRNAs was analyzed in both the GEO {"type":"entrez-geo","attrs":{"text":"GSE51908","term_id":"51908"}}GSE51908 and the {"type":"entrez-geo","attrs":{"text":"GSE142700","term_id":"142700"}}GSE142700 databases. miR-1287-5p expression was lower in AML patients compared with controls in both the {"type":"entrez-geo","attrs":{"text":"GSE51908","term_id":"51908"}}GSE51908 and the {"type":"entrez-geo","attrs":{"text":"GSE142700","term_id":"142700"}}GSE142700 datasets. Moreover, we demonstrated that miR-1287-5p expression was down-regulated in AML patients and up-regulated in circ_0059707-overexpressing cells. Collectively, our research demonstrated that the down-regulation of circ_0059707 was highly evident in de novo AML patients. Our analysis also demonstrated that circ_0059707 inhibited cell growth and promoted apoptosis by up-regulating miR-1287-5p.     

Dual inhibition of TGF‐β and PD‐L1: a novel approach to cancer treatment

Transforming growth factor‐β (TGF‐β) and programmed death ligand 1 (PD‐L1) initiate signaling pathways with complementary, nonredundant immunosuppressive functions in the tumor microenvironment (TME). In the TME, dysregulated TGF‐β signaling suppresses antitumor immunity and promotes cancer fibrosis, epithelial‐to‐mesenchymal transition, and angiogenesis. Meanwhile, PD‐L1 expression inactivates cytotoxic T cells and restricts immunosurveillance in the TME. Anti‐PD‐L1 therapies have been approved for the treatment of various cancers, but TGF‐β signaling in the TME is associated with resistance to these therapies. In this review, we discuss the importance of the TGF‐β and PD‐L1 pathways in cancer, as well as clinical strategies using combination therapies that block these pathways separately or approaches with dual‐targeting agents (bispecific and bifunctional immunotherapies) that may block them simultaneously. Currently, the furthest developed dual‐targeting agent is bintrafusp alfa. This drug is a first‐in‐class bifunctional fusion protein that consists of the extracellular domain of the TGF‐βRII receptor (a TGF‐β ‘trap’) fused to a human immunoglobulin G1 (IgG1) monoclonal antibody blocking PD‐L1. Given the immunosuppressive effects of the TGF‐β and PD‐L1 pathways within the TME, colocalized and simultaneous inhibition of these pathways may potentially improve clinical activity and reduce toxicity.