Reduced Tumor Cell Proliferation Research Articles

Ketoconazole and Posaconazole Selectively Target HK2-expressing Glioblastoma Cells

Hexokinase II (HK2) protein expression is elevated in glioblastoma (GBM), and we have shown that HK2 could serve as an effective therapeutic target for GBM. Here, we interrogated compounds that target HK2 effectively and restrict tumor growth in cell lines, patient-derived glioma stem cells (GSCs), and mouse models of GBM.Experimental Design: We performed a screen using a set of 15 drugs that were predicted to inhibit the HK2-associated gene signature. We next determined the EC50 of the compounds by treating glioma cell lines and GSCs. Selected compounds showing significant impact in vitro were used to treat mice and examine their effect on survival and tumor characteristics. The effect of compounds on the metabolic activity in glioma cells was also assessed in vitro. This screen identified the azole class of antifungals as inhibitors of tumor metabolism. Among the compounds tested, ketoconazole and posaconazole displayed the greatest inhibitory effect on GBM both in vitro and in vivo. Treatment of mice bearing GBM with ketoconazole and posaconazole increased their survival, reduced tumor cell proliferation, and decreased tumor metabolism. In addition, treatment with azoles resulted in increased proportion of apoptotic cells. Overall, we provide evidence that azoles exert their effect by targeting genes and pathways regulated by HK2. These findings shed light on the action of azoles in GBM. Combined with existing literature and preclinical results, these data support the value of repurposing azoles in GBM clinical trials.

Nutrition and Cancercapsaicin Treatment Reduces Tumor Growth, Tumor Cell Proliferation Ex Vivo and Partially Reverses Cancer Cachexia in Walker 256 Tumor-Bearing Rats

Capsaicin (trans-8-methyl-n-vanillyl-6-nonenamide) is the main pungent component found in hot peppers.Aim: In this study, we investigated the effect of capsaicin treatment on tumor growth and the metabolic indicators of cachexia in Walker 256 tumor-bearing rats.Methods: Male Wistar rats were inoculated subcutaneously in the right flank with 1 ml of a sterile suspension of 3 × 107 Walker tumor cells. The treated groups received capsaicin intraperitoneal 5 mg/kg body weight for 13 days.Results: The tumor weight on Day 14 in the non-treated group was 18 g. The rats also had a body weight loss, hypoglycemia, hyperlactacidemia, hypertriacylglycerolemia, and a depletion in glycogen storage. Treatment with capsaicin decreased tumor growth by 49% and a reversal of triacylglycerol serum. We also found a 32% reduction in tumor cell proliferation ex vivo. Lactate serum concentrations and body weight were lower but did not reach control levels.Conclusion: The treatment with capsaicin reduces tumor growth and cellular proliferation along with increased apoptosis and partial cachexia reversal.

The Potential Role of Medicinal Mushrooms in the Prevention and Treatment of Gynecological Cancers: A Review.

A review of scientific information about the potential role of medicinal mushrooms in the prevention and treatment of gynecological cancers, human immunodeficiency virus, and human papillomavirus infections is reported here. The results of in vivo and in vitro experiments on 16 different species of Basidiomycetes and three Ascomycetes, which possess chemopreventive potential and are effective in clinical application in combination with chemotherapy, are also discussed. Medicinal mushroom extracts confirm an evident efficacy on the reduction of tumor cell proliferation and side effects in patients with gynecological tumors who are undergoing chemotherapy treatments. This review, the first on the use of medicinal mushrooms in the prevention and treatment of gynecological cancers, aims to highlight the remarkable potential of mushrooms in integrated oncology.

Augmentation of Nab-Paclitaxel Chemotherapy Response by Mechanistically Diverse Antiangiogenic Agents in Preclinical Gastric Cancer Models.

Gastric adenocarcinoma (GAC) remains the third most common cause of cancer-related deaths worldwide. Systemic chemotherapy is commonly recommended as a fundamental treatment for metastatic GAC; however, standard treatment has not been established yet. Angiogenesis plays a crucial role in the progression and metastasis of GAC. We evaluated therapeutic benefits of mechanistically diverse antiangiogenic agents in combination with nab-paclitaxel, a next-generation taxane, in preclinical models of GAC. Murine survival studies were performed in peritoneal dissemination models, whereas tumor growth studies were performed in subcutaneous GAC cell-derived or patient-derived xenografts. The mechanistic evaluation involved IHC and Immunoblot analysis in tumor samples. Nab-paclitaxel increased animal survival that was further improved by the addition of antiangiogenic agents ramucirumab (or its murine version DC101), cabozantinib and nintedanib. Nab-paclitaxel combination with nintedanib was most effective in improving animal survival, always greater than 300% over control. In cell-derived subcutaneous xenografts, nab-paclitaxel reduced tumor growth while all three antiangiogenic agents enhanced this effect, with nintedanib demonstrating the greatest inhibition. Furthermore, in GAC patient-derived xenografts the combination of nab-paclitaxel and nintedanib reduced tumor growth over single agents alone. Tumor tissue analysis revealed that ramucirumab and cabozantinib only reduced tumor vasculature, whereas nintedanib in addition significantly reduced tumor cell proliferation and increased apoptosis. Effects of nab-paclitaxel, a promising chemotherapeutic agent for GAC, can be enhanced by new-generation antiangiogenic agents, especially nintedanib. The data suggest that nab-paclitaxel combinations with multitargeted antiangiogenic agents carry promising potential for improving clinical GAC therapy. Mol Cancer Ther; 17(11); 2353-64. ©2018 AACR.

The adaptive regulation of thiamine pyrophosphokinase-1 facilitates malignant growth during supplemental thiamine conditions.

Supplemental levels of vitamin B1 (thiamine) have been implicated in tumor progression. Tumor cells adaptively up-regulate thiamine transport during hypoxic stress. Upon uptake, thiamine pyrophosphokinase-1 (TPK1) facilitates the rapid phosphorylation of thiamine into thiamine pyrophosphate (TPP). However, the regulation of TPK1 during hypoxic stress is undefined. Understanding how thiamine homeostasis changes during hypoxia will provide critical insight into the malignant advantage supplemental thiamine may provide cancer cells. Using Western blot analysis and RT-PCR, we have demonstrated the post-transcriptional up-regulation of TPK1 in cancer cells following hypoxic exposure. TPK1 expression was also adaptively up-regulated following alterations of redox status by chemotherapeutic and antioxidant treatments. Although TPK1 was functionally up-regulated by hypoxia, HPLC analysis revealed a reduction in intracellular TPP levels. This loss was reversed by treatment with cell-permeable antioxidants and corresponded with reduced ROS production and enhanced cellular proliferation during supplemental thiamine conditions. siRNA-mediated knockdown of TPK1 directly enhanced basal ROS levels and reduced tumor cell proliferation. These findings suggest that the adaptive regulation of TPK1 may be an essential component in the cellular response to oxidative stress, and that during supplemental thiamine conditions its expression may be exploited by tumor cells for a redox advantage contributing to tumor progression.

Tumoral response and tumoral phenotypic changes in a rat model of diethylnitrosamine-induced hepatocellular carcinoma after salirasib and sorafenib administration.

BackgroundSeveral intracellular signaling pathways that are deregulated during hepatocarcinogenesis might constitute potential targets for hepatocellular carcinoma (HCC) therapy. The aim of this study was to test the potential synergic antitumor effect of salirasib and sorafenib in a diethylnitrosamine (DEN)-induced HCC model in rat. The hypothesis of tumor phenotype changes during treatment was also analyzed.Materials and methodsDEN was administered to Wistar rats during 9 weeks to induce cirrhosis and liver cancer. After tumor development, rats were treated with intraperitoneal injections of dimethyl sulfoxide (DMSO), or salirasib, and/or with oral sorafenib 5 days/week, during 4 weeks. At sacrifice, number and size of liver tumors as well as tumor burden were recorded, and all liver tumors were processed for histological and immunohistological analyses.ResultsMortality rate was significantly higher in rats treated with salirasib and/or sorafenib than in the control group (P=0.001). Tumor burden was smaller in the treated group compared with the DMSO control group (P=0.044), but a synergistic effect of the two chemotherapies could not be observed. In 62.5% of rats (10/16) treated with salirasib and/or sorafenib, a cytokeratin-7 and -19-positive hepatocholangiocellular carcinoma (HCC/CHC) was found vs 20% (5/25) developing such phenotype in the DMSO control group (P=0.018). Ki67 immunostaining showed significantly reduced tumor cell proliferation in treated rats (P=0.001), whereas apoptosis as assessed by caspase-3 activity in cell lysate was similar in all groups.ConclusionsThe addition of sorafenib to salirasib did not seem to provide any synergistic therapeutic effect in this study. Both chemotherapeutic agents, administered alone or in combination, induced tumoral phenotypic changes in the majority of rats, a finding not associated with an increased tumor cell proliferation or decreased apoptosis. The rat model described in this work constitutes the first experimental tool generating putatively more aggressive combined HCC/CHC tumors following chemotherapy. Further work is required to better characterize this clinically relevant phenomenon.

The Protein Tyrosine Phosphatase Activity of Eyes Absent Contributes to Tumor Angiogenesis and Tumor Growth.

DNA damage repair capacity is required for cells to survive catastrophic DNA damage and proliferate under conditions of intratumoral stress. The ability of the minor histone protein H2AX to serve as a hub for the assembly of a productive DNA damage repair complex is a necessary step in preventing DNA damage-induced cell death. The Eyes Absent (EYA) proteins dephosphorylate the terminal tyrosine residue of H2AX, thus permitting assembly of a productive DNA repair complex. Here, we use genetic and chemical biology approaches to separately query the roles of host vascular endothelial cell and tumor cell EYA in tumor growth. Deletion of Eya3 in host endothelial cells significantly reduced tumor angiogenesis and limited tumor growth in xenografts. Deletion of Eya3 in tumor cells reduced tumor cell proliferation and tumor growth without affecting tumor angiogenesis. A chemical inhibitor of the EYA tyrosine phosphatase activity inhibited both tumor angiogenesis and tumor growth. Simultaneously targeting the tumor vasculature and tumor cells is an attractive therapeutic strategy because it could counter the development of the more aggressive phenotype known to emerge from conventional antiangiogenic agents. Mol Cancer Ther; 17(8); 1659-69. ©2018 AACR.

Abstract 5429: Targeting Nrf2 anti-oxidative pathway as a novel strategy to suppress IDH1-mutated cancer

Abstract Background: Reactive oxygen species are recently recognized as a second messenger that affects many aspects in cell biology. In cancer cells, ROS has been proof closely related to metabolic reprogramming, tumor proliferation, progression and metastasis. In tumors with isocitrate dehydrogenase (IDH) mutations, the neomorphic enzyme results in unusual accumulation of ROS in cancer cells, suggesting distinctive vulnerabilities may be established in these types of cancer. However, the biologic function of ROS in IDH1-mutated cancers remains elusive. Methods: In this study, we explored the ROS homeostasis in IDH1 mutated glioma. We investigated the correlation between ROS scavenging genes and overall survival in 530 IDH1-mutated glioma cases. Mechanistically, we focused on nuclear factor-erythroid 2-related factor 2 (Nrf2) pathway and seek for novel therapeutic strategy by targeting ROS burden in cancer cells. We further investigated the therapeutic value of Nrf2 inhibitor, Brusatol, in IDH1 mutated cancer in vitro and in vivo. Results: We found IDH1 mutation leads to substantial reprogramming in ROS homeostasis, highlighted with prompted ROS generation and detoxification. Stronger expression of ROS scavenging gene predicts drug resistance and poor disease outcome. Enhanced Nrf2-associated scavenging pathway was found essential to maintain reasonable intracellular ROS level during cancer progression. Targeting Nrf2 scavenging pathway not only led to ROS overload, DNA/protein oxidative damage, but also reduced tumor cell proliferation and xenograft growth in vivo. Conclusions: IDH1 mutant glioma highly depend on Nrf2 scavenging pathway to maintain ROS homeostasis. Targeting ROS scavenging might be a novel approach to treat gliomas with IDH1 mutation. Citation Format: Yang Liu. Targeting Nrf2 anti-oxidative pathway as a novel strategy to suppress IDH1-mutated cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5429.

Abstract 2502: Role of TMEM97/σ2 receptor in breast cancer cell proliferation and responses to chemotherapy

Abstract Introduction: Breast cancer is the most prevalent cancer for women both in the developed and the developing countries. In 2017, about 40,610 women are expected to die from breast cancer in the U.S. The sigma receptors (σRs) are transmembrane receptors with two subtypes, termed sigma-1 receptor and sigma-2 receptor. Sigma-2 receptor has been found overexpressed in numerous human cancer tissues, and compound targeting it are now in clinical trials for breast cancer diagnosis. But it's biological role in breast cancer is unknown because the encoded gene has not been clearly identified. Recently, it has been demonstrated that TMEM97, not progesterone Receptor Membrane Component 1 (PGRMC1), is a bona fide sigma-2 receptor. The identification of sigma-2 (σ2) receptor will make it possible to define its biological functions in breast cancer and develop modulator of σ2 receptor as potential cancer therapeutics. Methods: The expression of TMEM97/σ2 receptor in multiple cancer cells was evaluated by RT-qPCR. Regulation of P53 on TMEM97/σ2 receptor was determined by Western Blot after 48 hours Doxorubicin treatment of MCF7 cell and HCT116 wild type and P53-/- cells. PB28, an agonist of σ2 receptor and an antagonist of σ1 receptor, was used (0, 1μM, 10μM and 20μM for 4 days) to test its effects on MCF7 cell growth and colony formation. TMEM97/σ2 receptor was cloned and sequenced. We overexpressed TMEM97/σ2 receptor in MCF7 cells to characterize its role in cell proliferation, survival, and colony formation, with or without treatment of its agonist PB28. Results and conclusion: TMEM97/σ2 receptor is expressed in various cancer cell lines including lung, breast and prostate cancer, albeit at different levels. TMEM97 expression was reduced in MCF7 cells when p53 expression was stimulated by doxorubicin. PB28 reduced the number of viable cells and also reduced colony formation of MCF7 cells in a dose dependent manner. Meanwhile overexpression of TMEM97/σ2 receptor retarded cell growth in MCF7 cells and sensitized them to PB28 as evidenced by reduced IC50 when compared with control cells. The data suggest that TMEM97/σ2 receptor is expressed in breast cancer cells, with its expression suppressed by p53, and activation of this receptor led to reduced tumor cell proliferation. Citation Format: Yuanqin Zhang, Jiuhui Wang, Daotai Nie. Role of TMEM97/σ2 receptor in breast cancer cell proliferation and responses to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2502.

Inhibition of Microsomal Prostaglandin E Synthase-1 in Cancer-Associated Fibroblasts Suppresses Neuroblastoma Tumor Growth

Despite recent progress in diagnosis and treatment, survival for children with high-risk metastatic neuroblastoma is still poor. Prostaglandin E2 (PGE2)-driven inflammation promotes tumor growth, immune suppression, angiogenesis and resistance to established cancer therapies. In neuroblastoma, cancer-associated fibroblasts (CAFs) residing in the tumor microenvironment are the primary source of PGE2. However, clinical targeting of PGE2 with current non-steroidal anti-inflammatory drugs or cyclooxygenase inhibitors has been limited due to risk of adverse side effects. By specifically targeting microsomal prostaglandin E synthase-1 (mPGES-1) activity with a small molecule inhibitor we could block CAF-derived PGE2 production leading to reduced tumor growth, impaired angiogenesis, inhibited CAF migration and infiltration, reduced tumor cell proliferation and a favorable shift in the M1/M2 macrophage ratio. In this study, we provide proof-of-principle of the benefits of targeting mPGES-1 in neuroblastoma, applicable to a wide variety of tumors. This non-toxic single drug treatment targeting infiltrating stromal cells opens up for combination treatment options with established cancer therapies.

Nigericin Exerts Anticancer Effects on Human Colorectal Cancer Cells by Inhibiting Wnt/β-catenin Signaling Pathway.

Nigericin, an antibiotic derived from Streptomyces hygroscopicus, which works by acting as an H+, K+, and Pb2+ ionophore, has exhibited promising anticancer activity. The main purpose of this study is to investigate its inhibitory effects on Wnt/β-catenin signaling pathway in colorectal cancer cells and clarify the underlying mechanism. We exposed two colorectal cancer lines (SW620 and KM12) to increasing concentrations of nigericin for different time periods and the 50% inhibiting concentration (IC50) values were evaluated. Our data showed that nigericin treatment significantly reduced tumor cell proliferation in dose- and time-dependent manners in colorectal cancer cells. The subsequent experiments in vitro and in vivo implied that nigericin could significantly suppress the tumor growth, migration, and invasion, and induce the apoptosis of colorectal cancer cells. Our results of Western blot and immunofluorescence assay showed that nigericin could suppress the Wnt/β-catenin signaling pathway in colorectal cancer cells with dose-dependent increased expressions of downstream effectors and target proteins. To further elucidate the inhibitory effects of nigericin via a β-catenin-dependent signaling mechanism, we established the stably β-catenin overexpression colorectal cancer cells. Western blot, SuperTOPFlash luciferase reporter, and immunoprecipitation assays all confirmed β-catenin as a critical intermediary and player in Wnt/β-catenin pathway, and nigericin exerted anticancer effects on colorectal cancer cells by directly targeting the β-catenin destruction complex. These results suggested that Wnt/β-catenin signaling might have an essential role in colorectal cancer progression. Nigericin targeting Wnt/β-catenin signaling might provide new insight into the molecular mechanism of nigericin toward cancer cells, and suggest possible clinical application in colorectal cancer. Mol Cancer Ther; 17(5); 952-65. ©2018 AACR.

Avicularin reversed multidrug-resistance in human gastric cancer through enhancing Bax and BOK expressions

5-Fluorouracil (5-Fu) and cisplatin (DDP) as important therapies in treatment of human gastric cancer have been widely determined. However, the therapeutic effects are usually hampered due to drug resistance or toxicity at high concentrations for application. Avicularin (AL, quercetin-3-α-l-arabinofuranoside), a bio-active flavonol isolated from a number of plants, has been reported to display diverse pharmacological properties. In this study, we explored the hypothesis by which AL reversed 5-Fu or DDP resistance in gastric cancer and the underlying molecular mechanism. Here, in vitro, the drug-resistant cancer cells were incubated to AL or DDP alone or the combination of AL and DDP. Then, MTT, colony formation, Hoechst 33258, flow cytometry and western blot analysis were used to investigate the effects of AL in the regulation of drug-resistance gastric cancer cells. The results indicated that AL treatment markedly re-sensitizes the drug resistant cells (SGC-7901/5-Fu and SGC-7901/DDP) to cytotoxicity of 5-Fu or DDP. Molecular mechanism analysis indicated that AL and DDP combination treatment enhanced apoptosis in SGC-7901/DDP cells, accompanied with the up-regulation of cleaved Caspase-3 and PARP, as well as the activation of pro-apoptotic signals, including Bax and BOK. Significantly, down regulation of Bax or BOK expressions using Bax siRNA or BOK siRNA decreased the inhibitory role of DDP in apoptosis of SGC-7901/DDP cells pretreated with AL, demonstrating that AL-reversed DDP resistance was associated with Bax and BOK expression. In vivo, AL and DDP combination significantly reduced gastric tumor growth. Immunohistochemical analysis indicated that co-treatment of AL and DDP significantly induced apoptosis, and reduced tumor cell proliferation in tumor tissue samples. Furthermore, we also found that the Bax, BOK, cleaved Caspase-3 and PARP expression in tumor tissues were highly induced by AL and DDP co-treatment. Together, our findings may provide a novel combination therapeutic strategy in treatment of human gastric cancer.

IN VIVO ANTITUMOR ACTIVITY OF PHYTOCHEMICAL PITC-2 OBTAINED FROM TISSUE CULTURED PLANT PLUCHEA INDICA ON SARCOMA-180 SOLID TUMOR MICE MODEL

Objective: PITC-2 was isolated from the methanolic root extract of tissue cultured medicinal plant Pluchea indica (L.) Less. PITC-2 is a thiophene derivative which is 2-(Prop-1-ynyl)-5(5,6-dihydroxyhexa-1,3-diynyl)-thiophene. The main objective of the study is to evaluate the in vivo antitumor activity of PITC 2 against sarcoma-180 cancer cell in Swiss albino mice.Methods: The antitumor activity was evaluated by treatment with PITC-2 at a dose of 2.5 and 5 mg/kg b.w for 21 days on sarcoma-180 mice model. Cell viability was studied using 3-(4, 5- dimethylthiazol -2-yl)-2, 5-diphenyl tetrazolium bromide assay and cell apoptosis, G1 cell cycle arrest and reduction in tumor cell proliferation were evaluated by histopathological analysis and Bcl-2, cyclic-D1, and Ki-67 protein expression through immunohistochemistry study.Results: Precisely, PITC-2 had a cytotoxic effect on various in vitro cancer cells. Significant decreases in solid tumor volume and weight along with increase lifespan also observed. The histopathological and immunohistopathological examination indicates that PITC-2 induces apoptosis, typical morphological changes and suppresses tumor cell proliferation along with G1 cell cycle arrest through the downregulation of the intratumoral expression of Bcl-2, cyclic D1, and Ki-67 and thus highlighting antiproliferative and apoptotic properties against sarcoma-180 in vivo solid tumor model.Conclusion: The present results clearly demonstrate that PITC-2 significantly inhibits sarcoma-180 cell growth in a dose-dependent manner in in vivo mice model. Besides this, the study reveals a comprehensive perception of the possible mechanism behind the antitumor activity of PITC-2 by significant changes in the morphological, hematological, biochemical parameters in sarcoma-180 cells.

Pro-atherogenic high fat diet accelerates tumor growth in mice through activation of innate immunity

Inflammation is a physiologic protective process triggered in response to injury or infection. When unresolved, inflammation often promotes the development of chronic pathologies such as cancer or atherosclerosis. In atherosclerosis, hypercholesterolemia-associated monocytosis drives plaque formation in mice. Epidemiologic and experimental studies have shown the involvement of dyslipidiemia and low-grade inflammation on the development of multiple cancers. Surprisingly, the link between atherosclerosis and cancer has not been studied in details. The objective of our study was to question whether a pro-atherogenic high fat diet (HFD) would modify the development of solid tumors. Our experimental approach was to feed mice susceptible to atherosclerosis with a classical HFD (15% fat, 1.25% cholesterol) or a chow diet for 2 weeks before induction of tumor development. We monitored tumor development every two days and analyzed the mechanistic events by in vitro and in vivo experiments. We found that the proatherogenic HFD accelerated the growth of several solid tumors (B16F10 and TC1) in C57Bl6 mice, independently of cholesterol levels. In contrast, the HFD induced a significant increase of myeloid cells number in circulation and in the tumor microenvironment (flow cytometry). We isolated (Miltenyi) and analyzed the phenotype (qPCR and ELISA) of tumor-infiltrating leukocytes (TILs). We found a rapid upregulation of IL-1beta and VEGFa expression and a downregulation of several inflammatory cytokines. The supernatant of TILs isolated from mice under HFD and cultured for 48 hours, failed to reduce tumor cell proliferation in vitro (tritiated thymidine) and absence of IL-1beta, or selective depletion of neutrophils protected mice against tumor acceleration under HFD. These data show that in the context of a proaterogenic HFD, neutrophils accumulate in the tumor microenvironment and switch toward an immunosuppressive phenotype, thus promoting the fast growing of tumors.

Nanoliposome C6-Ceramide Increases the Anti-tumor Immune Response and Slows Growth of Liver Tumors in Mice

BACKGROUND & AIMSCeramide, a sphingolipid metabolite, affects T-cell signaling, induces apoptosis of cancer cells, and slows tumor growth in mice. However, it has not been used as a chemotherapeutic agent because of its cell impermeability and precipitation in aqueous solution. We developed a nanoliposome-loaded C6-ceremide (LipC6) to overcome this limitation and investigated its effects in mice with liver tumors.METHODSImmune competent C57BL/6 mice received intraperitoneal injections of carbon tetrachlo-ride and intra-splenic injections of oncogenic hepatocytes. As a result, tumors resembling human hepatocellular carcinomas developed in a fibrotic liver setting. After tumors formed, mice were given an injection of LipC6 or vehicle via tail vein every other day for 2 weeks. This was followed by administration, also via tail vein, of tumor antigen-specific (TAS) CD8+ T cells isolated from the spleens of line 416 mice, and subsequent immunization by intraperitoneal injection of tumor antigen-expressing B6/WT-19 cells. Tumor growth was monitored with magnetic resonance imaging. Tumor apoptosis, proliferation, and AKT expression were analyzed using immunohistochemistry and immunoblots. Cytokine production, phenotype, and function of TAS CD8+ T cells and tumor-associated macrophages (TAMs) were studied with flow cytometry, real-time polymerase chain reaction (PCR), and ELISA. Reactive oxygen species (ROS) in TAMs and bone marrow-derived macrophages, induced by colony stimulating factor 2 (GMCSF or CSF2) or colony stimulating factor 1 (MCSF or CSF1), were detected using a luminescent assay.RESULTSInjection of LipC6 slowed tumor growth by reducing tumor cell proliferation and phosphorylation of AKT, and increasing tumor cell apoptosis, compared with vehicle. Tumors grew more slowly in mice given the combination of LipC6 injection and TAS CD8+ T cells followed by immunization compared with mice given vehicle, LipC6, the T cells, or immunization alone. LipC6 injection also reduced numbers of TAMs and their production of ROS. LipC6 induced TAMs to differentiate into an M1 phenotype, which reduced immune suppression and increased activity of CD8+ T cells. These results were validated by experiments with bone marrow-derived macrophages induced by GMCSF or MCSF.CONCLUSIONSIn mice with liver tumors, injection of LipC6 reduces the number of TAMs and the ability of TAMs to suppress the anti-tumor immune response. LipC6 also increases the anti-tumor effects of TAS CD8+ T cells. LipC6 might therefore increase the efficacy of immune therapy in patients with hepatocellular carcinoma.

Blockade of insulin-like growth factors increases efficacy of paclitaxel in metastatic breast cancer

Breast cancer remains the leading cause of cancer death in women owing to metastasis and the development of resistance to established therapies. Macrophages are the most abundant immune cells in the breast tumor microenvironment and can both inhibit and support cancer progression. Thus, gaining a better understanding of how macrophages support cancer could lead to the development of more effective therapies. In this study, we find that breast cancer-associated macrophages express high levels of insulin-like growth factors 1 and 2 (IGFs) and are the main source of IGFs within both primary and metastatic tumors. In total, 75% of breast cancer patients show activation of insulin/IGF-1 receptor signaling and this correlates with increased macrophage infiltration and advanced tumor stage. In patients with invasive breast cancer, activation of Insulin/IGF-1 receptors increased to 87%. Blocking IGF in combination with paclitaxel, a chemotherapeutic agent commonly used to treat breast cancer, showed a significant reduction in tumor cell proliferation and lung metastasis in pre-clinical breast cancer models compared to paclitaxel monotherapy. Our findings provide the rationale for further developing the combination of paclitaxel with IGF blockers for the treatment of invasive breast cancer, and Insulin/IGF1R activation and IGF+ stroma cells as potential biomarker candidates for further evaluation.

Valproic acid reduces tumor cell survival and proliferation with inhibitors of downstream molecules of epidermal growth factor receptor pathway

Objectives: To evaluate the beneficial effect of treating tumor cells with valproic acid (VPA) in combination with inhibitors of various downstream molecules of epidermal growth factor receptor pathway for limiting tumor cell growth. Materials and Methods: Cytotoxic effect of VPA was tested with various combinations of inhibitors of PI3K-AKT, ERK1/2, Jun N-terminal kinases (JNK) as well as p38 kinases in A375 cells using methyl-thiazol-tetrazolium assay, clonogenic assay, and caspase assay. Antiproliferative effect of the combination was tested with ethynyl-2′-deoxyuridine incorporation assay. Results: Among the combinations tested, a combination of VPA with PI3K-AKT inhibitor showed enhanced tumor cell death and reduced tumor cell proliferation compared to the combination of VPA with ERK1/2, JNK, and p38 inhibitors at lower doses. Conclusions: Combination of VPA with PI3K-AKT inhibitor at lower concentration reduced tumor cell growth.

ETV4 collaborates with Wnt/β-catenin signaling to alter cell cycle activity and promote tumor aggressiveness in gastrointestinal stromal tumor.

Gastrointestinal stromal tumor (GIST) is the most common sarcoma, often resulting from a KIT or platelet-derived growth factor receptor alpha (PDGFRA) mutation. The lineage transcription factor ETV1 is expressed similarly in GISTs regardless of malignant potential. Although the related transcription factor ETV4 has been associated with metastasis and tumor progression in other cancers, its role in GIST is unknown. In this study, we found that ETV4 levels were high in a subset of human GISTs and correlated with high mitotic rate. Through Gene Set Enrichment Analysis in selected human GISTs, we identified a relationship between ETV4 levels and β-catenin signaling, especially in advanced GISTs. GIST specimens with high ETV4 levels overexpressed cell cycle regulating genes and had aberrant activation of the canonical Wnt pathway. In human GIST cell lines, ETV4 RNA interference suppressed cell cycle genes and Wnt/β-catenin signaling. ETV4 knockdown also reduced tumor cell proliferation, invasion, and tumor growth in vivo. Conversely, ETV4 overexpression increased cyclin D1 expression and Wnt/β-catenin signaling. Moreover, we determined that ETV4 knockdown destabilized nuclear β-catenin and increased its degradation via COP1, an E3 ligase involved in both ETV4 and β-catenin turnover. Aberrant accumulation of ETV4 and nuclear β-catenin was found in patient derived xenografts created from metastatic GISTs that became resistant to tyrosine kinase inhibitors. Collectively, our findings highlight the significance of ETV4 expression in GIST and identify ETV4 as a biomarker in human GISTs.

ALK1Fc Suppresses the Human Prostate Cancer Growth in in Vitro and in Vivo Preclinical Models.

Prostate cancer is the second most common cancer in men and lethality is normally associated with the consequences of metastasis rather than the primary tumor. Therefore, targeting the molecular pathways that underlie dissemination of primary tumor cells and the formation of metastases has a great clinical value. Bone morphogenetic proteins (BMPs) play a critical role in tumor progression and this study focuses on the role of BMP9- Activin receptor-Like Kinase 1 and 2 (ALK1 and ALK2) axis in prostate cancer. In order to study the effect of BMP9 in vitro and in vivo on cancer cells and tumor growth, we used a soluble chimeric protein consisting of the ALK1 extracellular domain (ECD) fused to human Fc (ALK1Fc) that prevents binding of BMP9 to its cell surface receptors and thereby blocks its ability to activate downstream signaling. ALK1Fc sequesters BMP9 and the closely related BMP10 while preserving the activation of ALK1 and ALK2 through other ligands. We show that ALK1Fc acts in vitro to decrease BMP9-mediated signaling and proliferation of prostate cancer cells with tumor initiating and metastatic potential. In line with these observations, we demonstrate that ALK1Fc also reduces tumor cell proliferation and tumor growth in vivo in an orthotopic transplantation model, as well as in the human patient derived xenograft BM18. Furthermore, we also provide evidence for crosstalk between BMP9 and NOTCH and find that ALK1Fc inhibits NOTCH signaling in human prostate cancer cells and blocks the induction of the NOTCH target Aldehyde dehydrogenase member ALDH1A1, which is a clinically relevant marker associated with poor survival and advanced-stage prostate cancer. Our study provides the first demonstration that ALK1Fc inhibits prostate cancer progression, identifying BMP9 as a putative therapeutic target and ALK1Fc as a potential therapy. Altogether, these findings support the validity of ongoing clinical development of drugs blocking ALK1 and ALK2 receptor activity.

MiR-613 inhibits gastric cancer progression through repressing brain derived neurotrophic factor.

MicroRNA (miR)-613 has been reported to function as a tumor suppressor in several types of cancer. However, the biological function and underlying mechanism in gastric cancer (GC) has remained elusive. Therefore, the aim of the present study was to assess the expression and biological role of miR-613 in GC tissues and cell lines. miR-613 expression was found to be downregulated in 38 GC tissue samples compared to that in their adjacent non-cancerous tissues, and low expression of miR-613 was associated with lymph node metastasis and advanced tumor-nodes-metastasis stage. A gain-of-function assay demonstrated that miR-613 overexpression reduced tumor cell proliferation, migration and invasion of SGC-7901 cells, as determined by MTT and Transwell assays. Furthermore, brain-derived neutrophic factor (BDNF) was identified as a direct target of miR-613 in GC cells by a luciferase reporter assay. BDNF expression was upregulated and inversely correlated with miR-613 levels in GC tissues. In addition, knockdown of BDNF expression mimicked the tumor suppressive effect of miR-613 in GC cells. In conclusion, these findings demonstrated that miR-613 functions as a tumor suppressor in GC by targeting BDNF. Thus, miR-613 is a potential therapeutic target for GC.