Carcinogen Administration Research Articles

Response of CD68-Positive Thymic Cells in Rats to Selenium and Carcinogen Administration

Macrophages are the key thymic cells that take part in the antigen-independent differentiation of T-lymphocytes and their further selection. The level of macrophages indicates changes in the functions of the organ under study. As an essential trace element, selenium is part of a variety of proteins and enzymes, which perform cytoprotective, antimutagenic, and anticancerogenic action. Selenium also plays a significant role in the functioning of the immune system, increasing the activity of natural killers, production of interleukins, and stimulation of phagocytosis. This study was aimed at investigating the response of thymic macrophages to the administration of selenium and a chemical carcinogen. During the experiment, an increase in the level of CD68-positive cells was revealed in all experimental animals receiving selenium and a carcinogen, as well as under their combined action. The observed effects were assumed to be related to the immune-stimulating effect of selenium.

Реакция нейроэндокринных клеток и макрофагов селезенки на развитие опухоли в толстой кишке

Introduction. High cancer incidence requires finding new ways for comprehensive studying car-cinogenesis. Therefore, it is crucial to understand immune organ cell response and cell interaction in tumor development. The aim of the research was to study Synaptophysin+-, CD68+-cells, and biogenic amines in rat spleens during tumor development in the colon during dysplasia stages and adenocarcinoma formation. Materials and methods. Spleen histological slides of 110 mature male rats were studied 1 and 4 months after 1,2-dimethylhydrazine carcinogen administration using immunohistochemical, morphometric, and luminescent histochemical methods. Results. We found imbalanced production of biogenic amines (serotonin, histamine, and catecholamines) in the spleen and, therefore, a decrease in the cellular activity of the germinal centers of the lymphoid nodules. We also observed activation of periarteriolar lymphoid sheath (PALS) and red pulp in rats with precancerous colon lesions (1 month after carcinogen administration). At the same time, there was an increase in the number of CD68+ macrophages and Synaptophysin+ cells in the red pulp. In animals with adenocarcinoma (4 months after carcinogen introduction), the level of catecholamines in the luminescent granular cells of the PALS and the functional activity of these cells increased significantly. Simultaneously, the number of macrophages decreased in all the studied spleen compartments. Amid the decreased level of all biogenic amines in the red pulp, the quantity of Synaptophysin+ cells grew even more. Conclusion. The cells of all spleen compartments react to colon carcinogenesis, with reactivity of PALS cells and the red pulp being the most pronounced. The population of spleen macrophages undergoes rapid changes: their number increases in the red pulp in animals with precancerous lesions, while it decreases in all the splenic structures of rats with adenocarcinoma. Synaptophysin+ neuroendocrine cells of the red pulp play an important role in the reaction of the spleen to tumor development, and the number of these cells rises over time. Biogenic amines participate in the interaction of spleen cells with each other and with tumor-associated cells. Keywords: spleen, biogenic amines, neuroendocrine cells, synaptophysin, carcinogenesis

Repeated exposure of the oral mucosa over 12\xa0months with cold plasma is not carcinogenic in mice

Peri-implantitis may result in the loss of dental implants. Cold atmospheric pressure plasma (CAP) was suggested to promote re-osseointegration, decrease antimicrobial burden, and support wound healing. However, the long-term risk assessment of CAP treatment in the oral cavity has not been addressed. Treatment with two different CAP devices was compared against UV radiation, carcinogen administration, and untreated conditions over 12 months. Histological analysis of 406 animals revealed that repeated CAP exposure did not foster non-invasive lesions or squamous cell carcinoma (SCCs). Carcinogen administration promoted non-invasive lesions and SCCs. Molecular analysis by a qPCR screening of 144 transcripts revealed distinct inflammatory profiles associated with each treatment regimen. Interestingly, CAP treatment of carcinogen-challenged mucosa did not promote but instead left unchanged or reduced the proportion of non-invasive lesions and SCC formation. In conclusion, repeated CAP exposure of murine oral mucosa was well tolerated, and carcinogenic effects did not occur, motivating CAP applications in patients for dental and implant treatments in the future.

Chronic propranolol treatment moderately attenuated development of N-methyl-N-nitrosourea-induced mammary carcinoma in female rats.

The sympathetic nervous system participates in the development and progression of several cancer types and this effect is mediated mainly via β-adrenergic signaling. However, the potential of β-adrenergic signaling blockade to prevent cancer development after exposure to carcinogens has not been investigated, yet. Therefore, in our study, we determined the effect of the β-blocker propranolol on the development and progression of mammary cancer induced in female rats by administration of the chemical carcinogen N-methyl-N-nitrosourea (MNU). The propranolol treatment (20 mg/kg body weight) started 12 days after MNU administration and lasted 10 weeks. We found that both saline and propranolol treatment significantly increased gene expression of the catecholamine-synthesizing enzyme tyrosine hydroxylase, indicating that repeated injection of saline or propranolol-induced stress in these two groups. However, compared to the vehicle-treated group, propranolol slightly delayed the development and moderately reduced the incidence of mammary carcinoma in animals. To evaluate the mechanisms mediating the effect of propranolol on the development of MNU-induced cancer, we investigated several parameters of the tumor microenvironment and found that propranolol increased gene expression of Casp3. Our data indicate that propranolol treatment that starts after exposure to carcinogens might represent a new, useful approach for preventing the development of cancer, especially in stressed individuals. However, the potential efficiency of propranolol treatment for preventing cancer development and progression in individuals exposed to carcinogens needs further investigation.

Loss of PGRMC1 Delays the Progression of Hepatocellular Carcinoma via Suppression of Pro-Inflammatory Immune Responses.

Simple SummaryProgesterone receptor membrane component 1 (PGRMC1) and epidermal growth factor receptor (EGFR) are highly expressed in various cancers. Here, we first analyzed two sets of clinical data and found that the levels of PGRMC1 and EGFR in hepatocellular carcinomas (HCCs) were both inversely correlated with the survival of HCC patients. Accordingly, by using a carcinogen-induced mouse model of HCC, we found that Pgrmc1 knockout suppressed HCC development and extended the lifespan of HCC-bearing mice. In the acute setting of high-dose carcinogen administration, Pgrmc1 knockout was associated with increases in hepatic necrosis and decreases in the production of the pro-inflammatory cytokine IL-6. Indeed, silencing of Pgrmc1 in murine macrophages suppressed IL-6 production and NF-κB activity, and this process was significantly mediated by EGFR. Our study shows that Pgrmc1 affects the development of HCCs by regulating the EGFR-mediated inflammatory responses. Pgrmc1 may serve as a biomarker and a therapeutic target of HCC.Pgrmc1 is a non-canonical progesterone receptor related to the lethality of various types of cancer. PGRMC1 has been reported to exist in co-precipitated protein complexes with epidermal growth factor receptor (EGFR), which is considered a useful therapeutic target in hepatocellular carcinoma (HCC). Here, we investigated whether Pgrmc1 is involved in HCC progression. In clinical datasets, PGRMC1 transcription level was positively correlated with EGFR levels; importantly, PGRMC1 level was inversely correlated with the survival duration of HCC patients. In a diethylnitrosamine (DEN)-induced murine model of HCC, the global ablation of Pgrmc1 suppressed the development of HCC and prolonged the survival of HCC-bearing mice. We further found that increases in hepatocyte death and suppression of compensatory proliferation in the livers of DEN-injured Pgrmc1-null mice were concomitant with decreases in nuclear factor κB (NF-κB)-dependent production of interleukin-6 (IL-6). Indeed, silencing of Pgrmc1 in murine macrophages led to reductions in NF-κB activity and IL-6 production. We found that the anti-proinflammatory effect of Pgrmc1 loss was mediated by reductions in EGFR level and its effect was not observed after exposure of the EGFR inhibitor erlotinib. This study reveals a novel cooperative role of Pgrmc1 in supporting the EGFR-mediated development of hepatocellular carcinoma, implying that pharmacological suppression of Pgrmc1 may be a useful strategy in HCC treatment.

Tackling of Renal Carcinogenesis in Wistar Rats by Silybum marianum Total Extract, Silymarin, and Silibinin via Modulation of Oxidative Stress, Apoptosis, Nrf2, PPARγ, NF-κB, and PI3K/Akt Signaling Pathways.

The present work was designed to assess the efficacy of Silybum marianum total extract (STE), silymarin (Sm), and silibinin (Sb) against experimentally induced renal carcinogenesis in male Wistar rats and their roles in regulating oxidative stress, inflammation, apoptosis, and carcinogenesis. The diethylnitrosamine (DEN)/2-acetylaminofluorene (AAF)/carbon tetrachloride (CCl4)-administered rats were orally treated with STE (200 mg/kg b.w.), Sm (150 mg/kg b.w.), and Sb (5 mg/kg b.w.) every other day either from the 1st week or from the 16th week of carcinogen administration to the end of 25th week. The treatments with STE, Sm, and Sb attenuated markers of toxicity in serum, decreased kidney lipid peroxidation (LPO), and significantly reinforced the renal antioxidant armory. The biochemical results were further confirmed by the histopathological alterations. The treatments also led to suppression of proinflammatory mediators such as NF-κβ, p65, Iκβα, and IL-6 in association with inhibition of the PI3K/Akt pathway. Furthermore, they activated the expressions of PPARs, Nrf2, and IL-4 in addition to downregulation of apoptotic proteins p53 and caspase-3 and upregulation of antiapoptotic mediator Bcl-2. The obtained data supply potent proof for the efficacy of STE, Sm, and Sb to counteract renal carcinogenesis via alteration of varied molecular pathways.

Genetically engineered animal models of biliary tract cancers

Biliary tract cancers which include intrahepatic and extrahepatic cholangiocarcinomas and gallbladder cancer, are characterized by poor outcome. Therefore, identifying the molecular mechanisms of the disease has become a priority. However, such identification has to cope with extreme heterogeneity of the disease, which results from the variable anatomical location, the numerous cell types of origin and the high number of known genetic alterations. Animal models can develop invasive and metastatic tumours that recapitulate as faithfully as possible the molecular features of the human tumours. To generate animal models of cholangiocarcinoma, investigators resorted to the administration of carcinogens, induction of cholestasis, grafting of tumour cells and induction of genetic modifications. Here, we summarize the currently available genetically engineered animal models, and focus on mice and zebrafish. The experimental strategies that were selected to induce cholangiocarcinoma in a time-controlled and cell-type-specific manner are critically examined. We discuss their strengths and limitations while considering their relevance to human pathophysiology.

Ultrasonographic Follow-up of the Multistep Protocol for Prostate Cancer Induction in Wistar Rats.

This work intended to improve the knowledge of the rat model of prostate cancer (PCa) by ultrasonographic monitoring. Male Wistar rats were divided into control (n=8) and PCa (n=14) groups. PCa development was induced in the PCa group through the sequential administration of the anti-androgenic drug flutamide, testosterone propionate and the carcinogenic N-methyl-N-nitrosourea. The prostate was evaluated by ultrasonography at five timepoints along 49 weeks of the experimental protocol. Ventral prostate lobes were observed in all ultrasonographic examinations. The ventral prostate area of the control group increased gradually between the first and the last ultrasonographic examination. The ventral prostate area of PCa groups decreased due to flutamide administration and increased after androgen and carcinogen administration. The area of the dorsal prostate lobe increased between the fourth and the fifth ultrasonographic examination. In the last ultrasonographic examination, hypoechoic and anechoic lesions were observed in the PCa group. To our knowledge, this is the first study presenting a follow-up of rat prostatic dimensions by ultrasonography. Ultrasonography is a feasible approach for prostate cancer monitoring in experimental models.

Isomalto-oligosaccharides from rice and their potential use as pharma-nutraceuticals in prevention of colon cancer

Background: Isomalto-oligosaccharides (IMO) were enzymatically produced from rice (rIMO) alone or in combination with product (instant rice porridge mixed rIMO) demonstrated bifidogenic and butyrogenic effects in in vitro studies. The potential use of IMO from rice for pharma-nutraceuticals to prevent or risk reduction of colon cancer was further investigated in rats in this study.Objective: To investigate potential use of IMO from rice on prevention or risk reduction of colon cancer and gut microbiota modulation in rats, colonic polyp formation, histological changes, gut microbiota modulation, and butyrogenic properties were evaluated.Methods: An acute toxicity test was performed in ICR mice with a single dose of 2,000 mg/kg of tested IMO. The effects of rIMO were performed in AOM/DSS-induced Wistar rats. The animals were divided in 8 groups and treated three times a week with vehicle, three doses of rIMO, cIMO, and product (instant rice porridge mixed rIMO) before and after carcinogen administration. Animals were sacrificed at the end of 19th week.Results: Oral administration at up to 2,000 mg/kg of rIMO and product containing rIMO could be considered safe. Additionally, the animals chemically induced with AOM/DSS and treated with rIMO (1,500 mg/kg) and product containing rIMO significantly lower the occurrence of colonic polyps about 60%. There were no changes in other blood hematologic and biochemistry values but an improved gut barrier function when compared with animals in control and vehicle groups. AOM/DSS-induced rats supplemented with product containing rIMO could retard the reduction of beneficial bacteria and butyric acid production and thereby suppres the increase of harmful bacteria through the AOM/DSS post-induction phase.Conclusion: The results of this in vivo study suggest that consumption of rIMO alone or in combination with brown rice porridge could potentially help protect histological changes and risk reduction of developing for colon cancer. IMO from rice has potential use for pharma-nutraceuticals for risk reduction of colon cancer.Keywords: colon cancer, isomalto-oligosaccharides, prebiotic, rice, gut modulation

Abstract 645: Pregnancy inhibits mammary carcinogenesis by altering the mammary epithelial and stromal interactions

Abstract A full-term pregnancy before the age of 20 is the only known natural phenomenon that can drastically reduce the risk of breast cancer in women of all ethnic backgrounds worldwide. This universal protective effect of early pregnancy is clearly of major consideration in devising prevention strategies against breast cancer. It is well known that parous subjects have an altered systemic hormonal milieu with a significant decrease in circulating GH and PRL. Determining how these hormonal alterations impact the mammary gland of early parous rats is expected to improve our understanding of the dynamics of mammary carcinogenesis. The inguinal mammary fat pad was cleared of the host mammary gland in 3-4-week-old rats. One group of rats were exposed to N-Methyl-N-Nitrosourea (MNU) at 7 weeks of age, and another group of rats were not exposed to MNU. A subset of rats from each of these experimental groups were mated at 9 weeks and allowed to go through a full-term pregnancy. All animals were terminated 6 weeks post-weaning. Mammary epithelial cells (MECs) were isolated using the collagenase cell dissociation procedure. The isolated MECs (5 × 105) were transplanted into the gland-free fat pads of respective hosts. In brief, we studied the following groups to understand the influence of early parity on epithelial-stromal interactions involved in mammary carcinogenesis: 1) carcinogen treated MECs from Age-matched nulliparous (AMNP) rats were transplanted into untreated parous rats; 2) carcinogen treated MECs from parous rats were transplanted into untreated AMNP rats; 3) untreated MECs from AMNP rats were transplanted into carcinogen treated parous rats and 4) untreated MECs from parous rats were transplanted into carcinogen treated AMNP rats. All experimental animals were palpated weekly for the presence of mammary cancers. The cancerous nature of the palpable tumors was confirmed by histopathological analysis. Digital images of immunocytochemistry result was analyzed using Image Pro Plus software. Carcinogenesis experiments were terminated nine months after carcinogen administration. Incidence, multiplicity, and latency of mammary cancers was determined for each group and analyzed for significance by the appropriate statistical test. Our data demonstrated that transplantation of MECs from AMNP rats into parous hosts did not develop into palpable tumors while MECs from parous rats when transplanted to AMNP hosts developed mammary tumors. Analysis of the mammary gland wholemounts and molecular markers clearly indicated that parity alters key signaling mechanisms and inhibits progression of mammary lesions. These findings suggests that the mammary epithelial-stromal interactions in the parous rats is altered making it resistant to mammary carcinogenesis. Citation Format: Ramadevi Subramani Reddy, Adriana Galvez, Diego A. Pedroza, Animesh Chatterjee, Courtney Perry, Elizabeth Penner, Rajkumar Lakshmanaswamy. Pregnancy inhibits mammary carcinogenesis by altering the mammary epithelial and stromal interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 645.

Abstract 2730: Exceptional activity of ARN-509 (apalutamide) in prostate cancer prevention in rats

Abstract ARN-509 (apalutamide) is a “next-generation” non-steroidal androgen receptor (AR) antagonist that is approved by the FDA for treatment of non-metastatic castration-resistant prostate cancer. Studies were performed in a well-studied rat model to evaluate the efficacy of ARN-509 as an inhibitor of androgen-dependent prostate carcinogenesis. To support dose selection for the prostate cancer chemoprevention bioassay, a six-week pilot study was performed to evaluate the safety of ARN-509 in Wistar rats, and to characterize its PK parameters and dose-response relationships for pharmacodynamic activity (modulation of AR-dependent signaling pathways in the prostate). Several androgen-dependent signaling pathways were modulated by ARN-509; the most reliable biomarker of ARN-509 action in the prostate was down-regulation of kallikrein 1-related peptidase c9 (Klk1c9). Efficacy in prostate cancer prevention was evaluated using a rat model in which invasive prostate cancers are induced by sequential administration of anti-androgen, androgen, and chemical carcinogen (N-methyl-N-nitrosourea; MNU), followed by chronic androgen stimulation. Beginning one week after a single dose of MNU (30 mg/kg), male Wistar rats received daily oral (gavage) administration of ARN-509 at doses of 0 (vehicle control), 15, or 30 mg/kg body weight/day for 60 weeks; a fourth group received daily gavage administration of ARN-509 at 30 mg/kg/day for 60 weeks using an intermittent (one week on; one week off) schedule. Chronic oral administration of ARN-509 was well-tolerated by male Wistar rats; the compound is an extraordinarily potent inhibitor of prostate carcinogenesis. In comparison to a 53% incidence of accessory sex gland cancers (20/38 rats) and a 47% incidence of cancers that were clearly confined to the dorsolateral/anterior prostate (18/38 rats), cancer incidence in the accessory sex glands in both groups of rats receiving continuous daily administration of ARN-509 was 0% (30 mg/kg/day: 0/39 rats; 15 mg/kg/day: 0/37 rats). Intermittent (one week on; one week off) administration of the high dose of ARN-509 (30 mg/kg/day) was also highly effective in cancer prevention, but did not confer the complete protection seen with continuous daily administration: the group receiving intermittent exposure to ARN-509 demonstrated a 10% incidence of accessory sex gland cancer (4/39 rats), with 3% of animals (1/39 rats) demonstrating cancers that were clearly confined to the dorsolateral/anterior prostate. ARN-509 is the most potent inhibitor of prostate carcinogenesis identified in more than two dozen in vivo prostate cancer prevention bioassays performed in our laboratory, and is substantially more active in prostate cancer prevention in this model than is flutamide. These data suggest that ARN-509 merits consideration for evaluation in clinical trials for prostate cancer prevention. [Supported by HHSN261201500042I from NCI] Citation Format: Genoveva Murillo, Xinjian Peng, Miguel Muzzio, Maarten C. Bosland, Elizabeth R. Glaze, Chen Suen, David L. McCormick. Exceptional activity of ARN-509 (apalutamide) in prostate cancer prevention in rats [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2730.

Abstract 2824: Modulation of oral cancer cells survival and invasiveness by fusobacteria

Abstract Oral cancer represents a significant health burden worldwide while periodontitis is a common chronic infection of the periodontium. Many studies have suggested the association between periodontal disease and oral cancer. Studies have shown that biofilms on oral cancer lesions are rich in anaerobic bacteria, known to be associated with periodontal disease. This establishes that periodontal bacteria are present in the oral cancer microenvironment and likely directly interact with cancer cells. Therefore, it is vital to understand the interactions between the oral bacteria and cancer cells in order to improve treatment outcomes. The aim of this study was to investigate the effect of periodontal bacteria on oral cancer cells. Three oral cancer cell lines (OQ01, BHY and HN) were polyinfected with 4 bacteria associated with chronic periodontitis (Tanneralla forsynthia, Treponema denticola, Porphyromonas gingivalis and Fusobacterium nucleatum). Likely because BHY and HN already have a high baseline level of IL-8, OQ01 alone showed significantly enhanced IL-8 secretion (P< 0.05) but enhanced TFG-β secretion was detected in all cell lines tested. Polybacterial infection of oral cancer cells also upregulated mRNA for MMP1 (40 fold) and MMP9 (more than 60 fold), which are known to enhance cancer cell invasiveness. In addition, the expression of ZEB1 (4 fold higher), an oncogene known to induce epithelial mesenchymal transition in cancer cells, and MYC, JAK1 and STAT3, oncogenes involved in cell survival, were all significantly enhanced in polybacterial infected cancer cells. Further analysis using OQ01 cells in a single bacterial infection showed that F. nucleatum alone had the same or greater effect (4 fold higher IL-8 secretion) as the polybacterial infection with all 4 bacteria. All three strains of F. nucleatum tested enhanced the invasiveness of the oral cancer cells in vitro. Interestingly, Fusobacterial supernatant alone was enough to induce the same invasive phenotype as live bacteria in OQ01. These results suggest that F. nucleatum is the main periodontal pathogen responsible for inducing an invasive phenotype in these oral cancer cells. For in vivo studies, we used a mouse model of infection-associated oral tumorigenesis, which included the induction of chronic periodontitis by P. gingivalis and F. nucleatum and the administration of an oral carcinogen 4-nitroquinoline-1-oxide (4NQO). The results demonstrated that infection with Fusobacteria and P. gingivalis promoted oral cancer progression and enhanced invasion compared to controls. Our data also showed that Fusobacteria enhanced both survival and invasiveness of oral cancer cells. Collectively, this is the first study revealing the interaction between oral cancer cells and multiple periodontal bacteria. The modulation of the tumor microenvironment by oral bacteria has the potential to identify target genes for cancer treatment and improve patients’ prognosis. Citation Format: Amani Harrandah, Sasanka S. Chukkapalli, William JR Dunn, Ann Progulske-Fox, Edward K. Chan. Modulation of oral cancer cells survival and invasiveness by fusobacteria [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2824.

Abstract 2405: B7H4 as a T cell inhibitory regulator in bladder cancer

Abstract Despite a high number of neoantigens and T cell infiltration, most locally advanced and metastatic bladder cancers (BC) (>70%) do not respond to anti-PD1 treatment. Thus, targeting additional checkpoints may play an important role in treatment of BC. We sought to identify negative regulators of T cell activity and evaluate their function using a previously validated pre-clinical carcinogen-induced mouse model of BC. To identify regulators of T cell activity, we first investigated the timing and infiltration of T cells during tumor development in the BBN mouse BC model. We found that numbers of CD4+ and CD8+ T cell increased dramatically within the first 1-2 weeks, but then decreased over the next six weeks decreasing to the lowest levels at 8 weeks before increasing again coordinating with increasing tumor volume. During the decrease in T cell numbers, macrophages increased and by transcriptome profiling we identified increased levels of the T cell inhibitory protein B7-H4. B7-H4+CD11b+ macrophages increased over 4 weeks with opposite expression of CD4+ and CD8+ T cells by FACS. By IHC we localized B7-H4 expression in macrophages adjacent to tumor. A bioinformatic analysis of the human TCGA identified highest B7-H4 expression in luminal infiltrated subtypes of MIBCs, but did not correlate with CD4, CD8 or overall tumor mutation burden. MIBCs with elevated B7-H4 expression had significantly worse survival. In vitro, addition of anti-B7-H4 significantly increased proliferation and IFN-γ production by human T cells co-cultured with B7-H4 expressing APCs. To determine the role of B7-H4 in BC, we treated mice with overt BC (beginning at 4 months of carcinogen administration) with vehicle, anti-PD1 or anti-B7-H4 antibody. Compared to vehicle treated animals, anti-B7-H4 treated mice had a reduction in the number of pT3 tumors (50% vs. 73%), but less than anti-PD1 treated tumors (27%). However, anti-B7-H4 treated mice had significantly increased tumor infiltrating CD8+ T cells per histological field (77.1+34.3 vs. 11.5+7.8; p=0.0121), as well as a significant increase splenic CD8+IFN-γ+ T cells and levels of secreted IFN-γ upon anti-CD3 stimulation. Transcriptome profiling of tumors that responded to anti-B7-H4 antibody by RNA-Seq identified a decrease in mitotic cell cycle, cell cycle checkpoints, RHO GTPases and mitotic spindle checkpoint pathways in responsive tumors. In summary, we confirm the expression of B7-H4 in macrophages found in a murine model of BC and correlate expression to poor survival in the TCGA of MIBC. Inhibition of B7-H4 results in lymphocyte proliferation in vitro and inhibition of B7-H4 in mice resulted in decreased tumor stage and increased CD8+ TILs. This data suggests that B7-H4 may be a candidate alternative checkpoint that can be targeted in humans with BC unresponsive to PD1 therapy. Citation Format: Joshua Meeks, Alexander P. Glaser, Damiano Fantini, Yanni Yu, Valerie Eaton, Joseph R. Podojil, Stephen D. Miller. B7H4 as a T cell inhibitory regulator in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2405.

Elevated Expression of A-Raf and FA2H in Hepatocellular Carcinoma is Associated with Lipid Metabolism Dysregulation and Cancer Progression.

Identification of events leading to hepatocellular carcinoma (HCC) progression is essential for understanding its pathophysiology. The aims of this study are to identify and characterize differentially expressed proteins in serum of HCC-bearing rats and the corresponding controls during cancer initiation, progression and tumorigenesis. Chemical carcinogens, N-Nitrosodiethylamine and 2-aminoacetylfluorine are administered to induce HCC to male Wistar rats. The 2D-Electrophoresis and PD-Quest analyses are performed to identify several differentially expressed proteins in serum of HCC-bearing animals. These proteins are further characterized by MALDI-TOF-MS/MS analyses. Using pathwaylinker a HCC-specific network is analyzed among the MALDITOF- MS/MS characterized proteins and their interactors. Carcinogen administration caused inflammation leading to liver injury and HCC development. Liver inflammation was confirmed by increase in the levels of TNF-α and IL-6 in carcinogen treated rats. We report significant increase in expression of two differentially expressed proteins, namely, A-Raf and Fatty Acid 2- Hydroxylase (FA2H), at early stage of HCC initiation, during its progression and at tumor stage. Real-time PCR analysis of mRNA for these proteins confirmed up-regulation of their transcripts. Further, we validated our experimental data with sera of clinically confirmed liver cancer patients. The study suggests that FA2H and A-Raf play a major role in the progression of HCC.

Cannabinoids decrease intestinal permeability and induce colonic CD103+ dendritic cells to increase T regulatory cells leading to decreased murine colitis-associated colon cancer

Abstract Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) are two abundant phytocannabinoids found in Cannabis sativa. Cannabinoids bind to cannabinoid receptors 1 and 2 (CB1 and CB2), located primarily on the neurons and immune cells, respectively. Cannabis and synthetic THC and CBD are quickly gaining attention for their therapeutic potential in diseases ranging from epilepsy to multiple sclerosis, and cancer pain. Colon cancer is the third leading cause of cancer-related deaths, and treatment options are limited to surgery, chemotherapy or radiation. Many physicians are now moving towards a strategy focused on prevention. Chronic inflammation is a cause of many cancers including colon cancer. Thus, we used a murine model of colitis induced colon cancer, wherein BL6 mice were given a single administration of carcinogen (azoxymethane) followed by DSS (1%) in the drinking water daily for 1 week followed by 2 weeks without DSS. This cycle, along with bi-weekly treatment with cannabinoids (CBD, THC, THC+CBD, or vehicle; all 10mg/kg; by oral gavage) continued for 10 weeks, as the progression of cancer was monitored. Our data revealed that treatment with THC and THC+CBD resulted in decreased polyp formation in mice with colon cancer. We also showed that cannabinoids increased CD103+ dendritic cells in the colonic lamina propria that led to an increase in the CD4+Nrp1+FoxP3+ T regulatory cells (nTregs), which was dependent on CB2. Cannabinoids also increased the colonic barrier integrity as assessed by increased levels of ZO-1, through CB1, and increased mucus production. These changes collectively demonstrated cannabinoid treatment reduced the incidence of colitis-associated colon cancer by inducing an anti-inflammatory response.

Estrogen Activation of G-Protein–Coupled Estrogen Receptor 1 Regulates Phosphoinositide 3-Kinase and mTOR Signaling to Promote Liver Growth in Zebrafish and Proliferation of Human Hepatocytes

Patients with cirrhosis are at high risk for hepatocellular carcinoma (HCC) and often have increased serum levels of estrogen. It is not clear how estrogen promotes hepatic growth. We investigated the effects of estrogen on hepatocyte proliferation during zebrafish development, liver regeneration, and carcinogenesis. We also studied human hepatocytes and liver tissues. Zebrafish were exposed to selective modifiers of estrogen signaling at larval and adult stages. Liver growth was assessed by gene expression, fluorescent imaging, and histologic analyses. We monitored liver regeneration after hepatocyte ablation and HCC development after administration of chemical carcinogens (dimethylbenzanthrazene). Proliferation of human hepatocytes was measured in a coculture system. We measured levels of G-protein-coupled estrogen receptor (GPER1) in HCC and nontumor liver tissues from 68 patients by immunohistochemistry. Exposure to 17β-estradiol (E2) increased proliferation of hepatocytes and liver volume and mass in larval and adult zebrafish. Chemical genetic and epistasis experiments showed that GPER1 mediates the effects of E2 via the phosphoinositide 3-kinase-protein kinase B-mechanistic target of rapamycin pathway: gper1-knockout and mtor-knockout zebrafish did not increase liver growth in response to E2. HCC samples from patients had increased levels of GPER1 compared with nontumor tissue samples; estrogen promoted proliferation of human primary hepatocytes. Estrogen accelerated hepatocarcinogenesis specifically in male zebrafish. Chemical inhibition or genetic loss of GPER1 significantly reduced tumor development in the zebrafish. In an analysis of zebrafish and human liver cells and tissues, we found GPER1 to be a hepatic estrogen sensor that regulates liver growth during development, regeneration, and tumorigenesis. Inhibitors of GPER1 might be developed for liver cancer prevention or treatment. The accession number in the Gene Expression Omnibus is GSE92544.

NADPH Oxidase 1 in Liver Macrophages Promotes Inflammation and Tumor Development in Mice

Although there are associations among oxidative stress, reduced nicotinamide adenine dinucleotide phosphate oxidase (NOX) activation, and hepatocellular carcinoma (HCC) development, it is not clear how NOX contributes to hepatocarcinogenesis. We studied the functions of different NOX proteins in mice after administration of a liver carcinogen. Fourteen-day-old Nox1-/- mice, Nox4-/- mice, Nox1-/-Nox4-/- (double-knockout) mice, and wild-type (WT) C57BL/6 mice were given a single intraperitoneal injection of diethylnitrosamine (DEN) and liver tumors were examined at 9 months. We also studied the effects of DEN in mice with disruption of Nox1 specifically in hepatocytes (Nox1ΔHep), hepatic stellate cells (Nox1ΔHep), or macrophages (Nox1ΔMac). Some mice were also given injections of the NOX1-specific inhibitor ML171. To study the acute effects of DEN, 8-12-week-old mice were given a single intraperitoneal injection, and liver and serum were collected at 72 hours. Liver tissues were analyzed by histologic examination, quantitative polymerase chain reaction, and immunoblots. Hepatocytes and macrophages were isolated from WT and knockout mice and analyzed by immunoblots. Nox4-/- mice and WT mice developed liver tumors within 9 months after administration of DEN, whereas Nox1-/- mice developed 80% fewer tumors, which were 50% smaller than those of WT mice. Nox1ΔHep and Nox1ΔHSC mice developed liver tumors of the same number and size as WT mice, whereas Nox1ΔMac developed fewer and smaller tumors, similar to Nox1-/- mice. After DEN injection, levels of tumor necrosis factor, interleukin 6 (IL6), and phosphorylated signal transducer and activator of transcription 3 were increased in livers from WT, but not Nox1-/- or Nox1ΔMac, mice. Conditioned medium from necrotic hepatocytes induced expression of NOX1 in cultured macrophages, followed by expression of tumor necrosis factor, IL6, and other inflammatory cytokines; this medium did not induce expression of IL6 or cytokines in Nox1ΔMac macrophages. WT mice given DEN followed by ML171 developed fewer and smaller liver tumors than mice given DEN followed by vehicle. In mice given injections of a liver carcinogen (DEN), expression of NOX1 by macrophages promotes hepatic tumorigenesis by inducing the production of inflammatory cytokines. We propose that upon liver injury, damage-associated molecular patterns released from dying hepatocytes activate liver macrophages to produce cytokines that promote tumor development. Strategies to block NOX1 or these cytokines might be developed to slow hepatocellular carcinoma progression.

Screening of Chemopreventive Agents in Animal Models: Results on Reproducibility, Agents of a Given Class, and Agents Tested During Tumor Progression.

Because of the importance of testing reproducibility of results, we present our findings regarding screening agents in preclinical chemoprevention studies in rodent models performed by the Chemopreventive Agent Development Research Group (CADRG) of the Division of Cancer Prevention of the NCI. These studies were performed via contracts to various commercial and academic laboratories. Primarily, results with positive agents are reported because positive agents may progress to the clinics. In testing reproducibility, a limited number of direct repeats of our standard screening assays were performed; which entailed initiating treatment shortly after carcinogen administration or in young transgenic mice and continuing treatment until the end of the study. However, three additional protocols were employed relating to reproducibility: (i) testing agents at lower doses to determine efficacy and reduced toxicity; (ii) testing agents later in tumor progression when microscopic lesions existed and, (iii) testing multiple agents of the same mechanistic class. Data with six models that were routinely employed are presented: MNU-induced ER-positive mammary cancer in rats; MMTV-Neu ER-negative mammary cancers in transgenic mice; AOM-induced colon tumors in rats; intestinal adenomas in Min mice; OH-BBN-induced invasive rat urinary bladder cancers in rats; and UV-induced skin squamous carcinomas in mice. It was found that strongly positive results were highly reproducible in the preclinical models evaluated. Cancer Prev Res; 11(10); 595-606. ©2018 AACR.

BINDING OF 3H-DMBA WITH BIOPOLYMERS OF MOTHER AND FETUS TISSUES IN RATS DURING TRANSPLACENTAL CARCINOGENESIS

The aim of the study was to establish regularities of activated 3H-7,12-dimetilbenz(a)antratcene (DMBA) translocation in the major biopolymers of target and non-target organs at the initiation stage of transplacental carcinogenesis. DMBA was administered i.p. to rats on the 21th day of the pregnancy. Analysis of liver and kidney tissues of mothers, as well as liver, kidney, and brain of offspring was performed at temporal points of 1 hr to 14 days after single carcinogen administration. A regular, but transient non-specific accumulation of DMBA-radioactivity in the lipid fraction mainly embryonic tissues was observed during the first 6 hrs after administration of the labeled carcinogen. By 6 hrs, the maximum increase of label in RNA of embryonic brain and kidney (target organ for tumor induction) was observed and then, from 6 to 12 hrs, an increase occurred in the number of radioactive DNA adducts of fetus that was maintained for 24 hrs in these tissues. Increased accumulation of label in proteins of embryonic kidney was recorded from 6 to 24 hrs, and brain - from 12 to 24 hrs after administration of 3H-DMBA and on the 7th day in proteins of these organs of newborn rats.

Liver antioxidant and aerobic status improves after metformin and melatonin administration in a rat model of high-fat diet and mammary carcinogenesis.

Oxidative stress is involved in the development of various cancers. In the present study, the effect of long-term administration of peroral antidiabetic metformin and pineal hormone melatonin on liver antioxidant and aerobic status in female Sprague-Dawley rats carrying mammary tumors induced by N-methyl-N-nitrosourea was evaluated. Both substances were administered in a preventive and curative manner (12 days before and 16 weeks after the carcinogen application). Carcinogen administration induced oxidative stress: the level of thiobarbituric acid reactive substances (TBARS) considered as a marker of reactive oxygen species (ROS) generation in liver increased as well as the level of oxidatively modified protein content (OMP; aldehyde and ketone derivates). Metformin administration restored succinate dehydrogenase and lactate dehydrogenase activity and associated ROS production and OMP content to the level of intact rats, with predominant activation of superoxide dismutase (SOD) and glutathione reductase (GR). Melatonin alone and in combination with metformin also decreased TBARS content. OMP content decreased in all groups receiving chemoprevention. The rise in total antioxidant capacity after melatonin and particularly metformin and melatonin combination might result from the initiation of anaerobic metabolism and increasing SOD, GR, and glutathione peroxidase activity. Long-term administration of metformin and melatonin exerts antioxidant properties in liver, especially in combination.