Knockdown of COPA,Identified by Loss-of-Function Screen,Induces Apoptosis and Suppresses Tumor Growth in Mesothelioma Mouse Model

Malignant mesothelioma is a highly aggressive tumor arising from serosal surfaces of the pleura and is triggered by past exposure to asbestos. Currently, there is no widely accepted treatment for mesothelioma. Development of effective drug treatments for human cancers requires identification of therapeutic molecular targets. We therefore conducted a large-scale functional screening of mesothelioma cells using a genome-wide small interfering RNA library. We determined that knockdown of 39 genes suppressed mesothelioma cell proliferation. At least seven of the 39 genes—COPA, COPB2, EIF3D, POLR2A, PSMA6, RBM8A, and RPL18A—would be involved in anti-apoptotic function. In particular, the COPA protein was highly expressed in some mesothelioma cell lines but not in a pleural mesothelial cell line. COPA knockdown induced apoptosis and suppressed tumor growth in a mesothelioma mouse model. Therefore, COPA may have the potential of a therapeutic target and a new diagnostic marker of mesothelioma.     

Cancer Associated Fibroblasts Promote Tumor Growth and Metastasis by Modulating the Tumor Immune Microenvironment in a 4T1 Murine Breast Cancer Model

Background

Local inflammation associated with solid tumors commonly results from factors released by tumor cells and the tumor stroma, and promotes tumor progression. Cancer associated fibroblasts comprise a majority of the cells found in tumor stroma and are appealing targets for cancer therapy. Here, our aim was to determine the efficacy of targeting cancer associated fibroblasts for the treatment of metastatic breast cancer.

Methodology/Principal Findings

We demonstrate that cancer associated fibroblasts are key modulators of immune polarization in the tumor microenvironment of a 4T1 murine model of metastatic breast cancer. Elimination of cancer associated fibroblasts in vivo by a DNA vaccine targeted to fibroblast activation protein results in a shift of the immune microenvironment from a Th2 to Th1 polarization. This shift is characterized by increased protein expression of IL-2 and IL-7, suppressed recruitment of tumor-associated macrophages, myeloid derived suppressor cells, T regulatory cells, and decreased tumor angiogenesis and lymphangiogenesis. Additionally, the vaccine improved anti-metastatic effects of doxorubicin chemotherapy and enhanced suppression of IL-6 and IL-4 protein expression while increasing recruitment of dendritic cells and CD8⁺ T cells. Treatment with the combination therapy also reduced tumor-associated Vegf, Pdgfc, and GM-CSF mRNA and protein expression.

Conclusions/Significance

Our findings demonstrate that cancer associated fibroblasts promote tumor growth and metastasis through their role as key modulators of immune polarization in the tumor microenvironment and are valid targets for therapy of metastatic breast cancer.     

CSTP1, a Novel Protein Phosphatase,Blocks Cell Cycle,Promotes Cell Apoptosis,and Suppresses Tumor Growth of Bladder Cancer by Directly Dephosphorylating Akt at Ser473 Site

Akt/protein kinase B is a pivotal component downstream of phosphatidylinositol 3-kinase (PI3K) pathway, whose activity regulates the balance between cell survival and apoptosis. Phosphorylation of Akt occurs at two key sites either at Thr308 site in the activation loop or at Ser473 site in the hydrophobic motif. The phosphorylated form of Akt (pAkt) is activated to promote cell survival. The mechanisms of pAkt dephosphorylation and how the signal transduction of Akt pathway is terminated are still largely unknown. In this study, we identified a novel protein phosphatase CSTP1(complete s transactivated protein 1), which interacts and dephosphorylates Akt specifically at Ser473 site in vivo and in vitro, blocks cell cycle progression and promotes cell apoptosis. The effects of CSTP1 on cell survival and cell cycle were abrogated by depletion of phosphatase domain of CSTP1 or by expression of a constitutively active form of Akt (S473D), suggesting Ser473 site of Akt as a primary cellular target of CSTP1. Expression profile analysis showed that CSTP1 expression is selectively down-regulated in non-invasive bladder cancer tissues and over-expression of CSTP1 suppressed the size of tumors in nude mice. Kaplan-Meier curves revealed that decreased expression of CSTP1 implicated significantly reduced recurrence-free survival in patients suffered from non-invasive bladder cancers.     

Tumor‐Targeting Salmonella typhimurium A1‐R Promotes Tumoricidal CD8+ T Cell Tumor Infiltration and Arrests Growth and Metastasis in a Syngeneic Pancreatic‐Cancer Orthotopic Mouse Model

The present study determined the effect of the tumor‐targeting strain Salmonella typhimurium A1‐R (S. typhimurium A1‐R) on CD8⁺ tumor‐infiltrating lymphocytes (TILs) in a syngeneic pancreatic‐cancer orthotopic mouse model. The effect of tumor‐targeting S. typhimurium A1‐R on CD8⁺ TILs was determined on the Pan02 murine pancreatic‐adenocarcinoma implanted orthotopically in the pancreatic tail of C57BL/6 immunocompromised mice. Three weeks after orthotopic implantation, mice were randomized as follows G1: untreated control group (n = 8); and G2: S. typhimurium A1‐R‐treatment group (n = 8, 1 × 10⁷ colony forming units [CFU]/body, iv, weekly, 3 weeks). On the 22nd day from initial treatment, all mice were sacrificed and tumors were harvested. The tumor‐volume ratio was defined as ratio of tumor volume on the 22nd day relative to the 1st day. The tumor volume ratio was significantly lower in the S. typhimurium A1‐R‐treated group (G2) (3.0 ± 2.8) than the untreated control (G1) (39.9 ± 30.7, P < 0.01). Hematoxylin and easin (H&E) staining on tumor sections was performed to evaluate tumor destruction which was classified according to the Evans grading system and found to be much greater in the S. typhimurium A1‐R‐treated mice (G2). Six mice in G1 had peritoneal dissemination, whereas no mice showed peritoneal dissemination in G2 (P < 0.01). Immunohistochemical staining with anti‐mouse CD8⁺ antibody was performed in order to detect TILs determined by calculating the average number of CD8⁺ cells in three high power fields (200×) in the treated and untreated tumors. The TIL score was significantly higher in G2 (133.5 ± 32.2) than G1 (45.1 ± 19.4, P < 0.001). The present study demonstrates that S. typhimurium A1‐R promotes CD8⁺ T cell infiltration and inhibition of tumor growth and metastasis. J. Cell. Biochem. 119: 634–639, 2018. © 2017 Wiley Periodicals, Inc.     

Soluble Urokinase Receptor Is Released Selectively by Glioblastoma Cells That Express Epidermal Growth Factor Receptor Variant III and Promotes Tumor Cell Migration and Invasion

Genomic heterogeneity is characteristic of glioblastoma (GBM). In many GBMs, the EGF receptor gene (EGFR) is amplified and may be truncated to generate a constitutively active form of the receptor called EGFRvIII. EGFR gene amplification and EGFRvIII are associated with GBM progression, even when only a small fraction of the tumor cells express EGFRvIII. In this study, we show that EGFRvIII-positive GBM cells express significantly increased levels of cellular urokinase receptor (uPAR) and release increased amounts of soluble uPAR (suPAR). When mice were xenografted with human EGFRvIII-expressing GBM cells, tumor-derived suPAR was detected in the plasma, and the level was significantly increased compared with that detected in plasma samples from control mice xenografted with EGFRvIII-negative GBM cells. suPAR also was increased in plasma from patients with EGFRvIII-positive GBMs. Purified suPAR was biologically active when added to cultures of EGFRvIII-negative GBM cells, activating cell signaling and promoting cell migration and invasion. suPAR did not significantly stimulate cell signaling or migration of EGFRvIII-positive cells, probably because cell signaling was already substantially activated in these cells. The activities of suPAR were replicated by conditioned medium (CM) from EGFRvIII-positive GBM cells. When the CM was preincubated with uPAR-neutralizing antibody or when uPAR gene expression was silenced in cells used to prepare CM, the activity of the CM was significantly attenuated. These results suggest that suPAR may function as an important paracrine signaling factor in EGFRvIII-positive GBMs, inducing an aggressive phenotype in tumor cells that are EGFRvIII-negative.     

miR-30b,Down-Regulated in Gastric Cancer,Promotes Apoptosis and Suppresses Tumor Growth by Targeting Plasminogen Activator Inhibitor-1

Background

Gastric cancer is one of the most common malignant diseases worldwide. Emerging evidence has shown that microRNAs (miRNAs) are associated with tumor development and progression. Our previous studies have revealed that H. pylori infection was able to induce the altered expression of miR-30b in gastric epithelial cells. However, little is known about the potential role of miR-30b in gastric cancer.

Methods

We analyzed the expression of miR-30b in gastric cancer cell lines and human gastric cancer tissues. We examined the effect of miR-30b mimics on the apoptosis of gastric cancer cells in vitro by flow cytometry (FCM) and caspase-3/7 activity assays. Nude mouse xenograft model was used to determine whether miR-30b is involved in tumorigenesis of gastric cancer. The target of miR-30b was identified by bioinformatics analysis, luciferase assay and Western blot. Finally, we performed the correlation analysis between miR-30b and its target expression in gastric cancer.

Results

miR-30b was significantly down-regulated in gastric cancer cells and human gastric cancer tissues. Enforced expression of miR-30b promoted the apoptosis of gastric cancer cells in vitro, and miR-30b could significantly inhibit tumorigenicity of gastric cancer by increasing the apoptosis proportion of cancer cells in vivo. Moreover, plasminogen activator inhibitor-1 (PAI-1) was identified as the potential target of miR-30b, and miR-30b level was inversely correlated with PAI-1 expression in gastric cancer. In addition, silencing of PAI-1 was able to phenocopy the effect of miR-30b overexpression on apoptosis regulation of cancer cells, and overexpression of PAI-1 could suppressed the effect of promoting cell apoptosis by miR-30b, indicating PAI-1 is potentially involved in miR-30b-induced apoptosis on cancer cells.

Conclusion

miR-30b may function as a novel tumor suppressor gene in gastric cancer by targeting PAI-1 and regulating the apoptosis of cancer cells. miR-30b could serve as a potential biomarker and therapeutic target against gastric cancer.     

Cell Line Derived from a Murine Sarcoma Virus (Moloney Pseudotype)-Induced Tumor: Cultural, Antigenic, and Virological Properties

A cell line derived from a murine sarcoma virus (Moloney pseudotype)-induced tumor has been established. It retains oncogenicity, releases both sarcoma and leukemia viruses, and has virus-induced cellular antigens.     

Suppression of Tumor Growth by Pleurotus ferulae Ethanol Extract through Induction of Cell Apoptosis,and Inhibition of Cell Proliferation and Migration

Cancer is the second leading cause of death worldwide. Edible medicinal mushrooms have been used in traditional medicine as regimes for cancer patients. Recently anti-cancer bioactive components from some mushrooms have been isolated and their anti-cancer effects have been tested. Pleurotus ferulae, a typical edible medicinal mushroom in Xinjiang China, has also been used to treat cancer patients in folk medicine. However, little studies have been reported on the anti-cancer components of Pleurotus ferulae. This study aims to extract bioactive components from Pleurotus ferulae and to investigate the anti-cancer effects of the extracts. We used ethanol to extract anti-cancer bioactive components enriched with terpenoids from Pleurotus ferulae. We tested the anti-tumour effects of ethanol extracts on the melanoma cell line B16F10, the human gastric cancer cell line BGC 823 and the immortalized human gastric epithelial mucosa cell line GES-1 in vitro and a murine melanoma model in vivo. Cell toxicity and cell proliferation were measured by MTT assays. Cell cycle progression, apoptosis, caspase 3 activity, mitochondrial membrane potential (MMP), migration and gene expression were studied in vitro. PFEC suppressed tumor cell growth, inhibited cell proliferation, arrested cells at G0/G1 phases and was not toxic to non-cancer cells. PFEC also induced cell apoptosis and necrosis, increased caspase 3 activity, reduced the MMP, prevented cell invasion and changed the expression of genes associated with apoptosis and the cell cycle. PFEC delayed tumor formation and reduced tumor growth in vivo. In conclusion, ethanol extracted components from Pleurotus ferulae exert anti-cancer effects through direct suppression of tumor cell growth and invasion, demonstrating its therapeutic potential in cancer treatment.     

Normalization of Tumor Microenvironment by Neem Leaf Glycoprotein Potentiates Effector T Cell Functions and Therapeutically Intervenes in the Growth of Mouse Sarcoma

We have observed restriction of the murine sarcoma growth by therapeutic intervention of neem leaf glycoprotein (NLGP). In order to evaluate the mechanism of tumor growth restriction, here, we have analyzed tumor microenvironment (TME) from sarcoma bearing mice with NLGP therapy (NLGP-TME, in comparison to PBS-TME). Analysis of cytokine milieu within TME revealed IL-10, TGFβ, IL-6 rich type 2 characters was switched to type 1 microenvironment with dominance of IFNγ secretion within NLGP-TME. Proportion of CD8⁺ T cells was increased within NLGP-TME and these T cells were protected from TME-induced anergy by NLGP, as indicated by higher expression of pNFAT and inhibit related downstream signaling. Moreover, low expression of FasR⁺ cells within CD8⁺ T cell population denotes prevention from activation induced cell death. Using CFSE as a probe, better migration of T cells was noted within TME from NLGP treated mice than PBS cohort. CD8⁺ T cells isolated from NLGP-TME exhibited greater cytotoxicity to sarcoma cells in vitro and these cells show higher expression of cytotoxicity related molecules, perforin and granzyme B. Adoptive transfer of NLGP-TME exposed T cells, but not PBS-TME exposed cells in mice, is able to significantly inhibit the growth of sarcoma in vivo. Such tumor growth inhibition by NLGP-TME exposed T cells was not observed when mice were depleted for CD8⁺ T cells. Accumulated evidences strongly suggest NLGP mediated normalization of TME allows T cells to perform optimally to inhibit the tumor growth.     

SD-208, a Novel Protein Kinase D Inhibitor,Blocks Prostate Cancer Cell Proliferation and Tumor Growth In Vivo by Inducing G2/M Cell Cycle Arrest

Protein kinase D (PKD) has been implicated in many aspects of tumorigenesis and progression, and is an emerging molecular target for the development of anticancer therapy. Despite recent advancement in the development of potent and selective PKD small molecule inhibitors, the availability of in vivo active PKD inhibitors remains sparse. In this study, we describe the discovery of a novel PKD small molecule inhibitor, SD-208, from a targeted kinase inhibitor library screen, and the synthesis of a series of analogs to probe the structure-activity relationship (SAR) vs. PKD1. SD-208 displayed a narrow SAR profile, was an ATP-competitive pan-PKD inhibitor with low nanomolar potency and was cell active. Targeted inhibition of PKD by SD-208 resulted in potent inhibition of cell proliferation, an effect that could be reversed by overexpressed PKD1 or PKD3. SD-208 also blocked prostate cancer cell survival and invasion, and arrested cells in the G2/M phase of the cell cycle. Mechanistically, SD-208-induced G2/M arrest was accompanied by an increase in levels of p21 in DU145 and PC3 cells as well as elevated phosphorylation of Cdc2 and Cdc25C in DU145 cells. Most importantly, SD-208 given orally for 24 days significantly abrogated the growth of PC3 subcutaneous tumor xenografts in nude mice, which was accompanied by reduced proliferation and increased apoptosis and decreased expression of PKD biomarkers including survivin and Bcl-xL. Our study has identified SD-208 as a novel efficacious PKD small molecule inhibitor, demonstrating the therapeutic potential of targeted inhibition of PKD for prostate cancer treatment.     

Olive Phenolics as c-Met Inhibitors: (-)-Oleocanthal Attenuates Cell Proliferation,Invasiveness, and Tumor Growth in Breast Cancer Models

Dysregulation of the Hepatocyte growth factor (HGF)/c-Met signaling axis upregulates diverse tumor cell functions, including cell proliferation, survival, scattering and motility, epithelial-to-mesenchymal transition (EMT), angiogenesis, invasion, and metastasis. (-)-Oleocanthal is a naturally occurring secoiridoid from extra-virgin olive oil, which showed antiproliferative and antimigratory activity against different cancer cell lines. The aim of this study was to characterize the intracellular mechanisms involved in mediating the anticancer effects of (-)-oleocanthal treatment and the potential involvement of c-Met receptor signaling components in breast cancer. Results showed that (-)-oleocanthal inhibits the growth of human breast cancer cell lines MDA-MB-231, MCF-7 and BT-474 while similar treatment doses were found to have no effect on normal human MCF10A cell growth. In addition, (-)-oleocanthal treatment caused a dose-dependent inhibition of HGF-induced cell migration, invasion and G1/S cell cycle progression in breast cancer cell lines. Moreover, (-)-oleocanthal treatment effects were found to be mediated via inhibition of HGF-induced c-Met activation and its downstream mitogenic signaling pathways. This growth inhibitory effect is associated with blockade of EMT and reduction in cellular motility. Further results from in vivo studies showed that (-)-oleocanthal treatment suppressed tumor cell growth in an orthotopic model of breast cancer in athymic nude mice. Collectively, the findings of this study suggest that (-)-oleocanthal is a promising dietary supplement lead with potential for therapeutic use to control malignancies with aberrant c-Met activity.     

Nuclear T3 Receptor : Depletion by Tunicamycin Despite the Absence of N-Linked Glycan Units in a Murine Preadipocyte Cell Line and Rat Liver

Abstract

In experiments designed to evaluate the possible presence of N-glycan units in the nuclear T3 receptor, tunicamycin markedly depleted the nuclear T3 receptor sites when added to the culture medium of the T3 responsive ob 17 preadipocyte cell line under conditions which almost totally abolished protein N-glycosylation without significant alteration of protein synthesis. The affinity for T3 was unchanged. However, no significant interaction could be detected between the T3 receptor solubilized from the ob 17 cells, or from rat liver, and several insolubilized lectins of different specificities. Furthermore, treatment of the cells with swainsonine, a Golgi mannosidase II inhibitor, did not lead to any significant interaction of the receptor with concanavalin A as it would have occurred if the receptor had contained complex glycan units unrecognized by this lectin. These results are strong arguments against the presence of N-glycosidic moieties in the nuclear T3 receptor from mouse adipose cells and rat liver. The receptor sites depletion after tunicamycin treatment may most probably reflect an indirect effect through other glycoproteins which could be on the one hand required for either receptor stabilization or localization in the chromatin, or on the other hand involved in receptor level regulation.     

The Role of Tumor Necrosis Factor in Host Defense against Scrub Typhus Rickettsiae. I. Inhibition of Growth of Rickettsia tsutsugamushi,Karp Strain,in Cultured Murine Embryonic Cells and Macrophages by Recombinant Tumor Necrosis Factor-Alpha

Recombinant murine tumor necrosis factor-alpha (TNF-α) inhibited intracellular growth of Rickettsia tsutsugamushi, Karp strain, in the mouse embryo cell line C3H/10T1/2 clone 8 at doses of 100 to 10 U/ml. The growth inhibitory effect of TNF-α was also evident when peritoneal exudate macrophages or bone marrow-derived macrophages were used as the host cell for rickettsial growth. Interferon-gamma (IFN-γ), at doses up to 1,000 U/ml, did not affect the growth of this strain of rickettsiae in the mouse embryo cell line but, as expected, profoundly inhibited rickettsial growth in peritoneal exudate macrophages and bone marrow-derived macrophages. The effect of TNF-α on rickettsial growth in the mouse embryo cell line was not reproducibly enhanced by IFN-γ. Treatment of the cell line with TNF-α delayed rickettsial cytopathic effects, but the rickettsiae ultimately grew to high numbers in the cells and caused cell death. These findings show that, at least in our system, R. tsutsugamushi is resistant to IFN-γ-mediated antirickettsial effects in cells other than macrophages. The results of this study support the suggestion that TNF-α may inhibit rickettsial growth in cells other than macrophages.     

Characterization of the Tumorlike (T) Antigen Induced by Type 12 Adenovirus : I. Purification of the Antigen from Infected KB Cells and a Hamster Tumor Cell Line

The T antigen induced by type 12 adenovirus was purified from KB cells infected in the presence of 10(-6)m 5-fluoro-2-deoxyuridine to inhibit synthesis of viral capsid antigens. The antigen was purified approximately 200-fold, and the purified product contained only negligible amounts of host-cell contaminants, as judged by the residual radioactivity from (14)C-labeled uninfected cells which had been added to infected cells at the initiation of the purification. Immunoelectrophoresis indicated that the purified T-antigen preparation contained a single antigenic species. The T antigen from a hamster cell line (HT-1) derived from a type 12 adenovirus-induced tumor was purified by the same procedure. The T antigens from the two different sources were shown to be immunologically similar by use of a rabbit antiserum prepared against the purified T antigen from infected KB cells and sera from hamsters bearing tumors induced by type 12 adenovirus.