Stratification of lncRNA modulation networks in breast cancer.

BACKGROUND: Recently, non-coding RNAs are of growing interest, and more scientists attach importance to research on their functions. Long non-coding RNAs (lncRNAs) are defined as non-protein coding transcripts longer than 200 nucleotides. We already knew that lncRNAs are related to cancers and will be dysregulated in them. But most of their functions are still left to further study. A mechanism of RNA regulation, known as competing endogenous RNAs (ceRNAs), has been proposed to explain the complex relationships among mRNAs and lncRNAs by competing for binding with shared microRNAs (miRNAs). METHODS: We proposed an analysis framework to construct the association networks among lncRNA, mRNA, and miRNAs based on their expression patterns and decipher their network modules. RESULTS: We collected a large-scale gene expression dataset of 1,061 samples from breast invasive carcinoma (BRCA) patients, each consisted of the expression profiles of 4,359 lncRNAs, 16,517 mRNAs, and 534 miRNAs, and applied the proposed analysis approach to interrogate them. We have uncovered the underlying ceRNA modules and the key modulatory lncRNAs for different subtypes of breast cancer. CONCLUSIONS: We proposed a modulatory analysis to infer the ceRNA effects among mRNAs and lncRNAs and performed functional analysis to reveal the plausible mechanisms of lncRNA modulation in the four breast cancer subtypes. Our results might provide new directions for breast cancer therapeutics and the proposed method could be readily applied to other diseases.

Modular signature of long non-coding RNA association networks as a prognostic biomarker in lung cancer.

BACKGROUND: Increasing amount of long non-coding RNAs (lncRNAs) have been found involving in many biological processes and played salient roles in cancers. However, up until recently, functions of most lncRNAs in lung cancer have not been fully discovered, particularly in the co-regulated lncRNAs. Thus, this study aims to investigate roles of lncRNA modules and uncover a module-based biomarker in lung adenocarcinoma (LUAD). RESULTS: We used gene expression profiles from The Cancer Genome Atlas (TCGA) to construct the lncRNA association networks, from which the highly-associated lncRNAs are connected as modules. It was found that the expression of some modules is significantly associated with patient's survival, including module N1 (HR = 0.62, 95% CI = 0.46-0.84, p = 0.00189); N2 (HR = 0.68, CI = 0.50-0.93, p = 0.00159); N4 (HR = 0.70, CI = 0.52-0.95, p = 0.0205) and P3 (HR = 0.68, CI = 0.50-0.92, p = 0.0123). The lncRNA signature consisting of these four prognosis-related modules, a 4-modular lncRNA signature, is associated with favourable prognosis in TCGA-LUAD (HR = 0.51, CI = 0.37-0.69, p value = 2.00e-05). Afterwards, to assess the performance of the generic modular signature as a prognostic biomarker, we computed the time-dependent area under the receiver operating characteristics (AUC) of this 4-modular lncRNA signature, which showed AUC equals 68.44% on 336th day. In terms of biological functions, these modules are correlated with several cancer hallmarks and pathways, including Myc targets, E2F targets, cell cycle, inflammation/immunity-related pathways, androgen/oestrogen response, KRAS signalling, DNA repair and epithelial-mesenchymal transition (EMT). CONCLUSION: Taken together, we identified four novel LUAD prognosis-related lncRNA modules, and assessed the performance of the 4-modular lncRNA signature being a prognostic biomarker. Functionally speaking, these modules involve in oncogenic hallmarks as well as pathways. The results unveiled the co-regulated lncRNAs in LUAD and may provide a framework for further lncRNA studies in lung cancer.

Identification of a gut microbiota member that ameliorates DSS-induced colitis in intestinal barrier enhanced Dusp6-deficient mice.

Strengthening the gut epithelial barrier is a potential strategy for management of gut microbiota-associated illnesses. Here, we demonstrate that dual-specificity phosphatase 6 (Dusp6) knockout enhances baseline colon barrier integrity and ameliorates dextran sulfate sodium (DSS)-induced colonic injury. DUSP6 mutation in Caco-2 cells enhances the epithelial feature and increases mitochondrial oxygen consumption, accompanied by altered glucose metabolism and decreased glycolysis. We find that Dusp6-knockout mice are more resistant to DSS-induced dysbiosis, and the cohousing and fecal microbiota transplantation experiments show that the gut/fecal microbiota derived from Dusp6-knockout mice also confers protection against colitis. Further culturomics and mono-colonialization experiments show that one gut microbiota member in the genus Duncaniella confers host protection from DSS-induced injury. We identify Dusp6 deficiency as beneficial for shaping the gut microbiota eubiosis necessary to protect against gut barrier-related diseases.

Dynamics of alternative polyadenylation in human preimplantation embryos.

Alternative polyadenylation (APA) affects the length of the 3' untranslated region (3'-UTR) and the regulation of microRNAs. Previous studies have shown that cancer cells tend to have shorter 3'-UTRs than normal cells. A plausible explanation for this is that it enables cancer cells to escape the regulation of microRNAs. Here, we extend this concept to an opposing context: changes in 3'-UTR length in the development of the human preimplantation embryo. Unlike cancer cells, during early development 3'-UTRs tended to become longer, and gene expression was negatively correlated with 3'-UTR length. Moreover, our functional enrichment results showed that length changes are part of the development mechanism. We also investigated the analogy of 3'-UTR length variation with respect to lncRNAs and found that, similarly, lncRNA length tended to increase during embryo development.

STT3-dependent PD-L1 accumulation on cancer stem cells promotes immune evasion.

Enriched PD-L1 expression in cancer stem-like cells (CSCs) contributes to CSC immune evasion. However, the mechanisms underlying PD-L1 enrichment in CSCs remain unclear. Here, we demonstrate that epithelial-mesenchymal transition (EMT) enriches PD-L1 in CSCs by the EMT/ß-catenin/STT3/PD-L1 signaling axis, in which EMT transcriptionally induces N-glycosyltransferase STT3 through ß-catenin, and subsequent STT3-dependent PD-L1 N-glycosylation stabilizes and upregulates PD-L1. The axis is also utilized by the general cancer cell population, but it has much more profound effect on CSCs as EMT induces more STT3 in CSCs than in non-CSCs. We further identify a non-canonical mesenchymal-epithelial transition (MET) activity of etoposide, which suppresses the EMT/ß-catenin/STT3/PD-L1 axis through TOP2B degradation-dependent nuclear ß-catenin reduction, leading to PD-L1 downregulation of CSCs and non-CSCs and sensitization of cancer cells to anti-Tim-3 therapy. Together, our results link MET to PD-L1 stabilization through glycosylation regulation and reveal it as a potential strategy to enhance cancer immunotherapy efficacy.

PARP Inhibitor Upregulates PD-L1 Expression and Enhances Cancer-Associated Immunosuppression.

Purpose: To explore whether a cross-talk exists between PARP inhibition and PD-L1/PD-1 immune checkpoint axis, and determine whether blockade of PD-L1/PD-1 potentiates PARP inhibitor (PARPi) in tumor suppression.Experimental Design: Breast cancer cell lines, xenograft tumors, and syngeneic tumors treated with PARPi were assessed for PD-L1 expression by immunoblotting, IHC, and FACS analyses. The phospho-kinase antibody array screen was used to explore the underlying mechanism of PARPi-induced PD-L1 upregulation. The therapeutic efficacy of PARPi alone, PD-L1 blockade alone, or their combination was tested in a syngeneic tumor model. The tumor-infiltrating lymphocytes and tumor cells isolated from syngeneic tumors were analyzed by CyTOF and FACS to evaluate the activity of antitumor immunity in the tumor microenvironment.Results: PARPi upregulated PD-L1 expression in breast cancer cell lines and animal models. Mechanistically, PARPi inactivated GSK3ß, which in turn enhanced PARPi-mediated PD-L1 upregulation. PARPi attenuated anticancer immunity via upregulation of PD-L1, and blockade of PD-L1 resensitized PARPi-treated cancer cells to T-cell killing. The combination of PARPi and anti-PD-L1 therapy compared with each agent alone significantly increased the therapeutic efficacy in vivoConclusions: Our study demonstrates a cross-talk between PARPi and tumor-associated immunosuppression and provides evidence to support the combination of PARPi and PD-L1 or PD-1 immune checkpoint blockade as a potential therapeutic approach to treat breast cancer. Clin Cancer Res; 23(14); 3711-20. ©2017 AACR.

Inhibition of VEGF165/VEGFR2-dependent signaling by LECT2 suppresses hepatocellular carcinoma angiogenesis.

Hepatocellular carcinoma (HCC) relies on angiogenesis for growth and metastasis. Leukocyte cell-derived chemotaxin 2 (LECT2) is a cytokine and preferentially expressed in the liver. Previous studies have found that LECT2 targets to both immune and tumor cells to suppress HCC development and vascular invasion. Although LECT2 did not affect HCC cells growth in vitro, it still suppressed HCC xenografts growth in immune-deficient mice, suggesting other cells such as stroma cells may also be targeted by LECT2. Here, we sought to determine the role of LECT2 in tumor angiogenesis in HCC patients. We found that LECT2 expression inhibited tumor growth via angiogenesis in the HCC xenograft model. Specifically, we demonstrated that recombinant human LECT2 protein selectively suppressed vascular endothelial growth factor (VEGF)165-induced endothelial cell proliferation, migration, and tube formation in vitro and in vivo. Mechanistically, LECT2 reduced VEGF receptor 2 tyrosine phosphorylation and its downstream extracellular signal-regulated kinase and AKT phosphorylation. Furthermore, LECT2 gene expression correlated negatively with angiogenesis in HCC patients. Taken together, our findings demonstrate that LECT2 inhibits VEGF165-induced HCC angiogenesis through directly binding to VEGFR2 and has broad applications in treating VEGF-mediated solid tumors.

Blocking c-Met-mediated PARP1 phosphorylation enhances anti-tumor effects of PARP inhibitors.

Poly (ADP-ribose) polymerase (PARP) inhibitors have emerged as promising therapeutics for many diseases, including cancer, in clinical trials. One PARP inhibitor, olaparib (Lynparza, AstraZeneca), was recently approved by the FDA to treat ovarian cancer with mutations in BRCA genes. BRCA1 and BRCA2 have essential roles in repairing DNA double-strand breaks, and a deficiency of BRCA proteins sensitizes cancer cells to PARP inhibition. Here we show that the receptor tyrosine kinase c-Met associates with and phosphorylates PARP1 at Tyr907 (PARP1 pTyr907 or pY907). PARP1 pY907 increases PARP1 enzymatic activity and reduces binding to a PARP inhibitor, thereby rendering cancer cells resistant to PARP inhibition. The combination of c-Met and PARP1 inhibitors synergized to suppress the growth of breast cancer cells in vitro and xenograft tumor models, and we observed similar synergistic effects in a lung cancer xenograft tumor model. These results suggest that the abundance of PARP1 pY907 may predict tumor resistance to PARP inhibitors, and that treatment with a combination of c-Met and PARP inhibitors may benefit patients whose tumors show high c-Met expression and who do not respond to PARP inhibition alone.

GIT1 promotes lung cancer cell metastasis through modulating Rac1/Cdc42 activity and is associated with poor prognosis.

G-protein-coupled receptor kinase interacting protein 1 (GIT1) is participated in cell movement activation, which is a fundamental process during tissue development and cancer progression. GIT1/PIX forming a functional protein complex that contributes to Rac1/Cdc42 activation, resulting in increasing cell mobility. Although the importance of Rac1/Cdc42 activation is well documented in cancer aggressiveness, the clinical importance of GIT1 remains largely unknown. Here, we investigated the clinical significance of GIT1 expression in non-small-cell lung cancer (NSCLC) and also verified the importance of GIT1-Rac1/Cdc42 axis in stimulating NSCLC cell mobility. The result indicated higher GIT1 expression patients had significantly poorer prognoses in disease-free survival (DFS) and overall survival (OS) compared with lower GIT1 expression patients. Higher GIT1 expression was an independent prognostic factor by multivariate analysis and associated with migration/invasion of NSCLC cells in transwell assay. In vivo studies indicated that GIT1 promotes metastasis of NSCLC cells. Finally, GIT1 was found to stimulate migration/invasion by altering the activity of Rac1/Cdc42 in NSCLC cells. Together, the GIT1 expression is associated with poor prognosis in patients with NSCLC. GIT1 is critical for the invasiveness of NSCLC cells through stimulating the activity of Rac1/Cdc42.

Dual targeting of tumor angiogenesis and chemotherapy by endostatin-cytosine deaminase-uracil phosphoribosyltransferase.

Several antiangiogenic drugs targeting VEGF/VEGF receptor (VEGFR) that were approved by the Food and Drug Administration for many cancer types, including colorectal and lung cancer, can effectively reduce tumor growth. However, targeting the VEGF signaling pathway will probably influence the normal function of endothelial cells in maintaining homeostasis and can cause unwanted adverse effects. Indeed, emerging experimental evidence suggests that VEGF-targeting therapy induced less tumor cell-specific cytotoxicity, allowing residual cells to become more resistant and eventually develop a more malignant phenotype. We report an antitumor therapeutic EndoCD fusion protein developed by linking endostatin (Endo) to cytosine deaminase and uracil phosphoribosyltransferase (CD). Specifically, Endo possesses tumor antiangiogenesis activity that targets tumor endothelial cells, followed by CD, which converts the nontoxic prodrug 5-fluorocytosine (5-FC) to the cytotoxic antitumor drug 5-fluorouracil (5-FU) in the local tumor area. Moreover, selective targeting of tumor sites allows an increasing local intratumoral concentration of 5-FU, thus providing high levels of cytotoxic activity. We showed that treatment with EndoCD plus 5-FC, compared with bevacizumab plus 5-FU treatment, significantly increased the 5-FU concentration around tumor sites and suppressed tumor growth and metastasis in human breast and colorectal orthotropic animal models. In addition, in contrast to treatment with bevacizumab/5-FU, EndoCD/5-FC did not induce cardiotoxicity leading to heart failure in mice after long-term treatment. Our results showed that, compared with currently used antiangiogenic drugs, EndoCD possesses potent anticancer activity with virtually no toxic effects and does not increase tumor invasion or metastasis. Together, these findings suggest that EndoCD/5-FC could become an alternative option for future antiangiogenesis therapy.

p53 regulates epithelial-mesenchymal transition and stem cell properties through modulating miRNAs.

The epithelial-mesenchymal transition (EMT) has recently been linked to stem cell phenotype. However, the molecular mechanism underlying EMT and regulation of stemness remains elusive. Here, using genomic approaches, we show that tumour suppressor p53 has a role in regulating both EMT and EMT-associated stem cell properties through transcriptional activation of the microRNA miR-200c. p53 transactivates miR-200c through direct binding to the miR-200c promoter. Loss of p53 in mammary epithelial cells leads to decreased expression of miR-200c and activates the EMT programme, accompanied by an increased mammary stem cell population. Re-expressing miR-200c suppresses genes that mediate EMT and stemness properties and thereby reverts the mesenchymal and stem-cell-like phenotype caused by loss of p53 to a differentiated epithelial cell phenotype. Furthermore, loss of p53 correlates with a decrease in the level of miR-200c, but an increase in the expression of EMT and stemness markers, and development of a high tumour grade in a cohort of breast tumours. This study elucidates a role for p53 in regulating EMT-MET (mesenchymal-epithelial transition) and stemness or differentiation plasticity, and reveals a potential therapeutic implication to suppress EMT-associated cancer stem cells through activation of the p53-miR-200c pathway.