Carcinogenesis Model Research Articles

4-nitroquinoline 1-oxide-induced oral epithelial lesions exhibit time- and stage-dependent changes in the tumor immune microenvironment.

Oral tongue squamous cell carcinoma (OTSCC) is the most common cancer of the oral cavity and is associated with high morbidity due to local invasion and lymph node metastasis. Tumor infiltrating lymphocytes (TILs) are associated with good prognosis in oral cancer patients and dictate response to treatment. Ectopic sites for immune activation in tumors, known as tertiary lymphoid structures (TLS), and tumor-associated high-endothelial venules (TA-HEVs), which are specialized lymphocyte recruiting vessels, are associated with a favorable prognosis in OSCC. Why only some tumors support the development of TLS and HEVs is poorly understood. In the current study we explored the infiltration of lymphocyte subsets and the development of TLS and HEVs in oral epithelial lesions using the 4-nitroquinoline 1-oxide (4NQO)-induced mouse model of oral carcinogenesis. We found that the immune response to 4NQO-induced oral epithelial lesions was dominated by T cell subsets. The number of T cells (CD4+, FoxP3+, and CD8+), B cells (B220+) and PNAd+ HEVs increased from the earliest to the latest endpoints. All the immune markers increased with the severity of the dysplasia, while the number of HEVs and B cells further increased in SCCs. HEVs were present already in early-stage lesions, while TLS did not develop at any timepoint. This suggests that the 4NQO model is applicable to study the dynamics of the tumor immune microenvironment at early phases of oral cancer development, including the regulation of TA-HEVs in OTSCC.

Analysis of Co-expression of HSP 70 and BCL 2 in Oral Precancer and Cancer Patients Using Immunohistochemistry.

Understanding of molecular model of oral carcinogenesis has carried cancer chemotherapy far forward from conventional drug therapies. Small molecule inhibitors have gained acceptance as it has fewer adverse effects and also provide targeted drug therapy. The association of HSP 70 (Heat Shock Protein 70) and BCL 2 (B-cell lymphoma 2) proteins with oral precancer and cancer is already established. However, the complex interaction between these two proteins and how they affect each other's expression is still not understood completely. In our study, we aimed to correlate the expression of HSP 70 and BCL 2 with different histopathological grades of oral precancer and cancer tissue samples using tissue immunohistochemistry. Tissue samples were taken from a total of 250 patients (100 OPMDs and 150 OSCCs) and subjected to immunohistochemistry using anti-human mouse monoclonal antibodies to HSP70 and BCL2. Immunostaining was done, and the immunostaining intensity distribution (IID) index was calculated. Immunoreactivity scores for both HSP 70 and BCL 2 correlated with different grades of dysplasia. However, only HSP 70 had a statistically significant association (p=0.066). We also found that HSP 70 showed an inverse correlation, with higher expression majorly seen in well-differentiated OSCCs. Our study unveiled the HSP 70-BCL 2 interaction and provides insights about how this might affect drug designing and help overcome therapeutic lags. However, further studies are needed to provide a comprehensive review of such interactions among various small molecules.

Conditional knockout mouse model reveals a critical role of peroxiredoxin 1 in oral leukoplakia carcinogenesis

Peroxiredoxin 1 (Prx1) is an antioxidant protein that may promote the carcinogenesis in oral leukoplakia (OLK). To investigate the effect of Prx1 on the oral mucosal epithelium of OLK, we generated a Prx1 conditional knockout (cKO) mouse model. The mRNA and gRNA were generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technique. An infusion cloning method was used to construct a homologous recombination vector. To obtain the F0 generation mice, fertilized eggs of C57BL/6J mice were microinjected with Cas9 mRNA, gRNA, and a donor vector. Polymerase chain reaction (PCR) amplification and sequencing were used to identify F1 generation mice. Using the cyclization recombination-enzyme-locus of the X-overP1 (Cre-loxP) system, we created a Prx1 cKO mouse model, and the effectiveness of the knockout was confirmed through immunohistochemistry. We examined the influence of Prx1 knockout on the occurrence of OLK in mice by constructing a model of tongue mucosa carcinogenesis induced by 4-nitroquinoline-1-oxide (4NQO). Prx1 modification was present in the F1 generation, as evidenced by PCR amplification and sequencing. Prx1flox/flox: Cre + mice exhibited normal growth and fertility. Immunohistochemical analysis revealed that tongue epithelial cells in Prx1flox/flox: Cre + mice displayed a distinct deletion of Prx1. An examination of the heart, liver, spleen, lung, and kidney tissues revealed no visible histological changes. Histological analysis showed a reduction in the occurrence of the malignant transformation of OLK in the tongue tissues of Prx1flox/flox: Cre + mice. Ki67 immunostaining showed that Prx1 knockout significantly inhibited cell proliferation in the tongue epithelial. Our research developed a conditional knockout mouse model for Prx1. The obtained results provide insights into the function of Prx1 in the development of oral cancer and emphasize its potential as a therapeutic target for precancerous oral lesions.

Human-mouse comparison of the multistage nature of radiation carcinogenesis in a mathematical model.

Mouse models are vital for assessing risk from environmental carcinogens, including ionizing radiation, yet the interspecies difference in the dose response precludes direct application of experimental evidence to humans. Herein, we take a mathematical approach to delineate the mechanism underlying the human-mouse difference in radiation-related cancer risk. We used a multistage carcinogenesis model assuming a mutational action of radiation to analyze previous data on cancer mortality in the Japanese atomic bomb survivors and in lifespan mouse experiments. Theoretically, the model predicted that exposure will chronologically shift the age-related increase in cancer risk forward by a period corresponding to the time in which the spontaneous mutational process generates the same mutational burden as that the exposure generates. This model appropriately fitted both human and mouse data and suggested a linear dose response for the time shift. The effect per dose decreased with increasing age at exposure similarly between humans and mice on a per-lifespan basis (0.72- and 0.71-fold, respectively, for every tenth lifetime). The time shift per dose was larger by two orders of magnitude in humans (7.8 and 0.046 years per Gy for humans and mice, respectively, when exposed at ~35% of their lifetime). The difference was mostly explained by the two orders of magnitude difference in spontaneous somatic mutation rates between the species plus the species-independent radiation-induced mutation rate. Thus, the findings delineate the mechanism underlying the interspecies difference in radiation-associated cancer mortality and may lead to the use of experimental evidence for risk prediction in humans.

Mitochondrial dysfunction of peripheral blood mononuclear cells is associated with lung carcinogenesis

The composition, quantity, and function of peripheral blood mononuclear cells (PBMCs) are closely correlated with tumorigenesis. However, the mechanisms of PBMCs in lung cancer are not clear. Mitochondria are energy factories of cells, and almost all cellular functions rely on their energy metabolism level. The present study aimed to test whether the mitochondrial function of PBMCs directly determines their tumor immune monitoring function. We recruited 211 subjects, including 105 healthy controls and 106 patients with recently diagnosed with lung cancer. The model of lung carcinogenesis induced by BaP was used in animal experiment, and the Bap carcinogenic metabolite, Benzo(a)pyren-7,8-dihydrodiol-9,10-epoxide (BPDE), was used in cell experiment. We found that mitochondrial function of PBMCs decreased significantly in patients with new lung cancer, regardless of age. In vivo, BaP caused PBMC mitochondrial dysfunction in mice before the appearance of visible malignant tissue. Moreover, mitochondrial function decreased significantly in mice with lung cancers induced by BaP compared to those without lung cancer after BaP intervention. In vitro, BPDE also induced mitochondrial dysfunction and reduced the aggressiveness of PBMCs toward cancer cells. Furthermore, the changes in mitochondrial energy metabolism gene expression caused by BPDE are involved in this process. Thus, the mitochondrial function of PBMCs is a potential prognostic biomarker or therapeutic target to improve clinical outcomes in patients with lung cancer.

In vivo model of colorectal cancer: impact to cigarette smoke exposition on neoplasm development

Our proposal aims to investigate the impact that exposure to cigarette smoke has on the development of lesions in the colorectal mucosa of in vivo model of colorectal carcinogenesis. For that, 24 young male rats were induced to colorectal carcinogenesis and separated into two groups: exposure to cigarette smoke for 20 weeks (DMH+/Tobacco+) and control (DMH+/Tobacco-). In the group (DMH+/Tobacco+), 14.7% dysplasia and 85.29% malignant neoplasms were observed, including tubular adenocarcinoma (73%), carcinoma in situ (17%) and signet-ring cell adenocarcinoma (10%). Mononuclear inflammation and pleomorphisms were mild to moderate. In the control group, there was 7.69% dysplasia, 7.69% tubular adenoma and 84.62% malignant neoplasms, including tubular adenocarcinoma (82%), signet-ring cell adenocarcinoma (9%), carcinoma in situ (5%) and mucinous adenocarcinoma (4%). Mononuclear inflammation and pleomorphisms were moderate to severe. Thus, the experimental model showed that the lesions were predominantly malignant and with histopathological characteristics compatible with human colorectal cancer, including its possible applications for future studies.

5-Methylcoumarin-4β-glucoside mitigated colon tumor progression in mice with AOM/DSS-induced colon carcinogenesis

ContextThe in vitro cytotoxicity profile of 5-Methylcoumarin-4β-glucoside (5-MC4βG) has been demonstrated to inhibit the proliferation of HT-29 adenocarcinoma cells. Aim: The need for newer and affordable chemotherapeutic agents is critical. Main methodsWe investigated the effect of 5-MC4βG on an azoxymethane /dextran sodium sulfate-induced colon carcinogenesis model in six groups of laboratory BALB/c mice for six weeks. While the first and second groups of mice served as vehicle and disease controls respectively, the third, fourth, and fifth groups were administered oral graded doses (25, 50, and 100 mg/kg) of 5-MC4βG. The sixth group was treated with 5-Flourouracil (15 mg/kg). Key findingsThe 100 mg/kg dose of 5-MC4βG upregulated APC mRNA expression by two-fold and downregulated β-catenin mRNA expression by two-fold compared to their respective disease controls. Furthermore, tumorigenic gene transcripts (MDM2, BCL2, CDK1, and cyclin D1) were downregulated by one-fold (except cyclin D1 which was downregulated by two-fold), whereas pro-apoptotic genes (p53, Bax, and Casp3) were upregulated by two-fold following treatment with 100 mg/kg dose of 5-MC4βG. At the metabolic level, the bioactive compound lowered the expression of classical tumor markers; tissue polypeptide antigen, tumor-associated glycoprotein 72, and carcinoembryonic antigen by at least half. Histologically, 5-MC4βG intervention revealed a reduction in neoplastic cells associated with cellular necrosis. Significance5-MC4βG reduced colon carcinogenesis in mice. Thus, this compound may be a promising candidate for colorectal cancer chemotherapy. Further development of 5-MC4βG will hopefully lead to the development of a potential anti-colon cancer drug candidate.

Abstract LB156: Targeting epigenetic activity of beta-catenin/CBP impedes oral cancer progression

Abstract Head and neck cancer is a complex malignancy with its major anatomical subsite, cancer of the oral cavity, ranking among the most deadly and disfiguring cancers. Oral cancer presents primarily as HPV-negative oral squamous cell carcinoma (OSCC) whose etiology is associated with tobacco and alcohol use and for which there are limited treatments. Whereas immunotherapies, such as anti-PD-1 antibodies, have become a promising therapeutic option, only ~20% of head neck cancer patients benefit from anti-PD-1 immunotherapy. We and others have shown that non-mutational epigenetic reprogramming mediated by the β-catenin/CBP complex promotes aggressive cell phenotypes in OSCC by establishing open chromatin at the transcription start sites of genes associated with cancer stemness. Accordingly, pharmacological inhibition of this complex dramatically inhibits OSCC evolution in a syngeneic mouse model of OSCC. Here, we evaluated whether the inhibition of β-catenin/CBP epigenetic activity would enhance the effects of anti-PD-1 immunotherapy. We adapted a syngeneic mouse model of tobacco-associated oral carcinogenesis that utilizes a mouse cell line, 4MOSC1, derived from 4-nitroquinoline-1 oxide- (4NQO)-induced tongue tumors of C57BL/6J mice. After tumors developed for 7 days, mice were blindly separated into treatment cohorts (n=13, each) to receive vehicle control (PBS), monotherapy (either 10 mg/kg anti-PD-1 EOD IP, or 100 mg/kg E7386, a Wnt signaling pathway modulator, QD oral gavage) or combination therapy (anti-PD-1 + E7386). Tumor isografts were harvested 11 days post initiation of treatments and processed for histopathology, immunohistochemistry, and immunofluorescence analyses. Measurements of tumor volumes showed that combination therapy resulted in a statistically significant reduction by ~80% compared to control mice and that it surpassed tumor decrease by either monotherapy. These results were confirmed by histopathology analyses. Evaluation of tumor tissues by immunofluorescence revealed statistically significant decreases in Cbp, H3K4me3, Keratin 14, and Podoplanin expression in response to E7386 alone and in combination with anti PD-1. Collectively our results suggest that a combination therapy comprising anti PD-1 treatment with E7386-targeting β-catenin/CBP epigenetic signaling is likely to impede OSCC evolution to advanced disease. Supported by Eisai, Co Ltd Research Award (MAK), NIH 5R01 DE030350 (MAK, SM, XV) and 5R01 DE031413 (MVB). Citation Format: Emily Fisher, Anthony Spinella, Junji Matsui, Kenichi Nomoto, Xaralabos Varelas, Manish Bais, Maria Kukuruzinska. Targeting epigenetic activity of beta-catenin/CBP impedes oral cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB156.

Abstract LB377: The combination of epigallocatechin gallate (EGCG) and resveratrol prevents 4-NQO-induced oral carcinogenesis in mice

Abstract Head and neck cancer (HNC), commonly known as oral cancer, is a devastating disease and the 6th most common cancer worldwide. Most HNC patients are diagnosed with advanced-stage disease for which the 5-year survival is below 50%, stressing the need for chemoprevention. Recently, we have reported that the combination of resveratrol and EGCG induces synergistic apoptosis and inhibits xenografted HNC growth by inhibiting the AKT-mTOR pathway. This study investigated the chemopreventive efficacy of resveratrol, EGCG, and their combination using the 4NQO-induced oral carcinogenesis model. C57BL/6 mice were exposed to 4-NQO (50 μg/ml) via drinking water for 10 weeks, followed by treatment with vehicle (50% sweetened condensed milk), resveratrol (30 mg/kg), EGCG (30 mg/kg) and their combination for 8 weeks, 5 days/week. The mice were sacrificed on week 24, and the number of visible and microscopic lesions was counted. Resveratrol alone and in combination with EGCG significantly inhibited the number of visible lesions. In contrast, the number of microscopic lesions and lesion area was significantly inhibited only in the combination group. The expression of Ki-67 was also significantly inhibited in resveratrol and combination groups when compared with untreated control. Furthermore, RNASeq and qPCR analysis using an HNC cell line identified GDF15, ATF3, p21, p27, and Bim as significantly upregulated genes, with GDF15 being the most upregulated one. Expression of GDF15 and ATF3 proteins was confirmed by Western blotting. Taken together, our data strongly demonstrate the chemopreventive potential of the combination of EGCG and resveratrol and pave the way for further clinical developments. (Supported by NIH Grants R15DE032063 and P20GM103434) Citation Format: Adeoluwa A. Adeluola, Lukmon Raji, Saroj Sigdel, ASM Anisuzzaman, A.R.M. Ruhul Amin. The combination of epigallocatechin gallate (EGCG) and resveratrol prevents 4-NQO-induced oral carcinogenesis in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB377.

Gedunin induces apoptosis and inhibits HMBG1/PI3K/AKT signaling pathways in a rat model of gastric carcinogenesis induced by methylnitronitrosoguanidine.

This study aimed to evaluate the anti-cancer effects of gedunin, a natural compound, in a rat model of gastric carcinogenesis induced by MNNG. Fifty-four rats were randomly assigned to six groups for a 60-day study on the effects of MNNG and gedunin. Groups 1-4 received 200 mg/kg MNNG (1, 10, or 100 mg/kg), and group 5 had only 100 mg/kg gedunin. Gedunin at low doses exhibited anti-cancer and protective properties against MNNG-induced damage, including reduced inflammation, and apoptosis. Gedunin demonstrates a U-shaped dose-response, with low doses offering protection and high doses promoting tumor growth.

Prenatal inflammation exposure accelerates lung cancer tumorigenesis in offspring mouse: possible links to IRE1α/XBP1-mediated M2-like polarization of TAMs and PD-L1 up-expression

BackgroundPrenatal inflammation exposure (PIE) can increase the disease susceptibility in offspring such as lung cancer. Our purpose was to investigate the mechanisms of PIE on lung cancer.MethodsPrenatal BALB/c mice were exposed to lipopolysaccharide (LPS), and then, their offspring were intraperitoneally instilled with urethane to establish the two-stage lung cancer carcinogenesis model. At the 48 weeks of age, the offspring mice were killed and lung tissues were collected for HE, immunohistochemistry, immunofluorescence, and Luminex MAGPIX®-based assays. CD11b + F4/80 + tumor-associated macrophages (TAMs) were sorted out from lung tumor tissues by cell sorting technique. Flow cytometry was employed to evaluate the extent of M2-like polarization of TAMs and PD-L1 expression.ResultsThe offspring of PIE mice revealed more lung lesion changes, including atypical hyperplasia and intrapulmonary metastases. The number of lung nodules, lung organ index, and PCNA, MMP-9 and Vimentin positive cells in lung tissue of PIE group were higher than those of Control group. The increases of mRNA encoding M2 macrophage markers and cytokines in offspring of prenatal LPS-treated mice confirmed the induced effect of PIE on macrophage polarization. Additionally, PIE treatment increased the percentage of CD163 + CD206 + cells in the sorted TAMs. Importantly, endoplasmic reticulum (ER) stress-markers like GRP78/BIP and CHOP, p-IRE1α and XBP1s, and PD-L1 were up-regulated in TAMs from PIE group. Besides, we also observed that IRE1α inhibitor (KIRA6) reversed the M2-like TAMs polarization and metastasis induced by PIE.ConclusionsIRE1α/XBP1-mediated M2-like TAMs polarization releases the pro-tumorigenic cytokines and PD-L1 expression, which may be the regulatory mechanism of accelerating lung cancer in offspring of mice undergoing PIE.

Different Oncogenes and Reproductive Histories Shape the Progression of Distinct Premalignant Clones in Multistage Mouse Breast Cancer Models

A remote carcinogen exposure can predispose to breast cancer onset decades later, suggesting carcinogen-induced mutations generate long-lived premalignant clones. How subsequent events influence the progression of specific premalignant clones remains poorly understood. Here, we generated multistage mouse models of mammary carcinogenesis by combining chemical carcinogen exposure (using 7,12-dimethylbenzanthracene, DMBA) with transgenes that enable inducible expression of one of two clinically relevant mammary oncogenes: c-MYC (MYC) or PIK3CAH1047R (PIK). In prior work, DMBA exposure generated mammary clones bearing signature HrasQ61L mutations, which only progressed to mammary cancer after inducible Wnt1 oncogene expression. Here, after an identical DMBA exposure, MYC versus PIK drove cancer progression from mammary clones bearing mutations in distinct Ras family paralogs. For example, MYC drove cancer progression from either Kras- or Nras-mutant clones, whereas PIK transformed Kras-mutant clones only. These Ras mutation patterns were maintained whether oncogenic transgenes were induced within days of DMBA exposure or months later. Completing a full-term pregnancy (parity) failed to protect against either MYC- or PIK-driven tumor progression. Instead, a postpartum increase in mammary tumor predisposition was observed in the context of PIK-driven progression. However, parity decreased the overall prevalence of tumors bearing Krasmut, and the magnitude of this decrease depended on both the number and timing of pregnancies. These multistage models may be useful for elucidating biological features of premalignant mammary neoplasia.

High fructose Corn Syrup recast glucose transporter-5, Wnt, NF-κB signalling and mitochondrial apoptosis in an animal model of oral oncogenesis

BackgroundWnt signalling pathway, is mediated by members of T-cell factor (TCF) transcription factors family, is essential for the control of epithelial cell proliferation and death. Glucose transporter-5 (GLUT5), fructose-specific transporter, is also important in allowing transcellular fructose uptake. The goal of this work to determine how the High fructose Corn Syrup (HFCS) affected Wingless-related integration site (Wnt) and Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signalling in the 7,12 –dimethylbenzaanthracene (DMBA)-induced hamster buccal pouch carcinogenesis (HBPCs) model. MethodsFour groups of hamsters were created. For 12 weeks, 0.5 % DMBA was applied 3 times/week to the left side buccal pouches of the hamsters in groups (2 & 4). Additionally, the animals in groups (3 & 4) were given through drinking water of HFCS 25 %. The control animals were from group 1. By using western blot analysis, signalling network markers of the GLUT-5, Wnt, TCF-4, GSK-3β and NF-κB as well as mitochondrial apoptotic pathway marker expression B-cell lymphoma protein 2 (Bcl-2)-associated X (Bax) and cyclooxygenase – 2 (COX-2) was assessed. ResultsDrinking water uptake of 25% HFCS encouraged progress of HBP carcinomas by constitutive stimulating of the Wnt pathway via GSK-3β overexpression. HFCS suppressed Wnt signalling which contributed the NF-κB attenuation and changes the signalling markers in apoptotic network. ConclusionsOur hypothesis suggests a mechanically crosstalk between Wnt and NF-κB signalling pathways in HBP carcinomas that is developed by HFCS. HFCS that targets the Wnt pathway and its downstream signalling mediators could be additive reason for cancer development.

Abstract 1466: Tobacco carcinogen impacts redox pathway to facilitate pancreatic ductal adenocarcinoma (PDAC) initiation and progression

Abstract The 5-year survival rate for pancreatic ductal adenocarcinoma (PDAC) is only 10%. PDAC is projected to soon become the second leading cause of cancer death. Tobacco is one of the main risk factors for PDAC. Incidence of PDAC increases 2-fold among tobacco users, and 20-35% of PDAC cases are attributed to smoking. However, the specific pathways activated by tobacco carcinogens remain elusive.This research project addresses the impact of carcinogens in pancreatic cancer etiology. We hypothesize that carcinogens in tobacco increase reactive oxygen species (ROS), which elevates the Nrf2 redox pathway to facilitate PDAC initiation and progression. Specifically, we focus on two major subclasses of carcinogens: the heavy metal cadmium (Cd) and the polycyclic aromatic hydrocarbon (PAH) benzo(a)pyrene (BaP). Using mouse pancreatic ductal organoid cultures, we found that acute carcinogen exposure increases Nrf2 activity, which is sustained after "intermediate" exposure (5 passages). Intriguingly, chronic exposure (10 passages) downregulates Nrf2 activity, which has been shown to be associated with acquired metastatic potential. Furthermore, we found an upregulation in NFkB and EGFR signaling after intermediate exposure, which are involved in microenvironment remodeling and increased proliferative potential. Overall, we demonstrate that acute carcinogen exposure elevates ROS levels and activates the Nrf2 pathway, whereas prolonged exposure upregulates NFkB and EGFR pathways. Furthermore, we have established a novel carcinogenesis model using 3D organoid culture to recapitulate each step in pancreatic tumorigenesis. Collectively, this project contributes to our understanding of tobacco-related PDAC pathogenesis. Citation Format: Xiaoxue Lin, McKenna Stamp, Alexandra Fowler, Chelsea Bottomley, Shira Okhovat, Kristina Peck, Dannielle Engle. Tobacco carcinogen impacts redox pathway to facilitate pancreatic ductal adenocarcinoma (PDAC) initiation and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1466.

Abstract 1464: Smoking carcinogen-induced inflammation promotes lung carcinogenesis via IRAK4 activation

Abstract Lung cancer is globally the most common cancer, and most cases are associated with smoking. Smoking exposes respiratory epithelial tissues to various carcinogens. Evaluating the effects of these carcinogens administered intratracheally in mouse models would allow us to study lung carcinogenesis in a relevant manner and determine therapeutically targetable pathways. Cigarette smoking exposure was mimicked in vivo using a smoke machine and the condensate was subjected to chemical analysis. In addition to nicotine, cigarette smoking condensate included significant levels of the carcinogens, Nicotine-derived nitrosamine ketone (NNK) and Benzo(α)pyrene (BP). Next, a mouse model of cigarette smoking-induced carcinogenesis was developed by exposing ICR mice to intratracheal exposure to NNK and BP three times a week for 18 months. Mice chronically exposed to NNK and BP developed epithelial dysplasia at 4 months of exposure and lung cancers at 8-12 mo. of exposure at significantly increased rates relative to controls. Histology revealed myeloid inflammation in murine lung tissues. Exposure of lung epithelial cells to cigarette smoking condensate led to increased production of pro-inflammatory IL-1β. Downstream mediator of IL-1β signaling, Interleukin 1 receptor associated kinase-4 (IRAK4), was overexpressed in murine lung tissues exposed to carcinogens in vivo. Two-thirds of human tissue sections obtained from archived lung cancers also exhibited overactivated IRAK4 expression. In lung cancer cell lines, IRAK4 immunofluorescence revealed a microtubule like pattern of expression in lung cancer cells. Immunoblotting confirmed IRAK4 colocalization within purified microtubules. Using mass spectrometry on isolated microtubules, we observed that phosphorylation decreased in various tubular proteins after IRAK4 inhibition. Inhibition of IRAK4 using small molecule specific inhibitors resulted in decreased invasion in lung cancer cell lines. These data show that chronic intratracheal exposure of smoking associated carcinogens leads to dysplasia and malignancies in mouse models. This exposure also leads to myeloid inflammation and activation of IRAK4 in lung tissues. Therapeutic targeting of IRAK4 can have potentially beneficial effects in lung cancer models. Citation Format: Ritesh K. Aggarwal, Simone Sidoli, Srabani Sahu, Srinivas Aluri, Charan Vegivinti, Divij Verma, Shanisha Gordon-Mitchell, Beamon Agarwal, Tanya Verma, Daniel T. Starczynowski, Ulrich G. Steidl, Balazs Halmos, Lindsay M. LaFave, Haiying Cheng, Amit Verma, Yiyu Zou. Smoking carcinogen-induced inflammation promotes lung carcinogenesis via IRAK4 activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1464.

Effect of inactivating heterozygous mutations in DNA repair genes on experimental lung carcinogenesis in mice

Background. Inactivating mutations in Chek2 and Gprc5a genes are known to be associated with cancer development. Experimental carcinogenesis studies in genetically modified mice generate new data on their influence on pathology development.Aim. In the present study in a model of lung carcinogenesis, survival parameters as well as tumor multiplicity and size in mice with Chek2 and Gprc5a heterozygous inactivating mutations were evaluated.Material and methods. F2 hybrid mice from crosses between CBAB6F1 males heterozygous for the studied mutations and wild-type BALB / c females were used: Chek2dAA-carriers (76 males and 64 females) and Gprc5ainsA-carriers (60 males and 42 females). Starting at four months of age, mice received urethane (ethyl carbamate) intraperitoneally at a dose of 600 mg / kg weekly for 6 weeks. After genotyping by allele-specific PCR, animals were allocated to groups. Carcinogenesis parameters were evaluated 40 weeks after the beginning of the experiment.Results. The proportion of mice with mutations surviving to the age of three months roughly followed the Mendelian distribution (35 / 41 males and 33 / 31 females) for the offspring of males heterozygous for Chek2dAA and was significantly lower in the case of Gprc5ainsA (20 / 40 males and 17 / 25 females, p = 0.043). The death of Gprc5ainsA carriers during the experiment was also higher than in the control group (p = 0.0506 in females). Synchronous lung and thymus neoplasms were found in 2 out of 4 Gprc5ainsA females that died before the end of the experiment, which were not found in other groups. At the end of the experiment, no significant differences in tumor multiplicity, mean linear size, and volume were found between the groups of mice with and without mutations.Conclusion. It was found that heterozygous inactivating mutation Chek2dAA does not affect early age development and does not modify the parameters of induced lung carcinogenesis in mice. Heterozygous carriage of Gprc5ainsA mutation in mice increases the risk of early death and sensitivity to the toxic and carcinogenic effects of urethane.

Abstract 7552: Inhibition of histone methyltransferase EZH2 for immune-interception of colorectal cancer in Lynch syndrome

Abstract Introduction: Colorectal pre-cancers in Lynch Syndrome (LS) display a distinct immune profile thus presenting a unique opportunity to develop novel immune-interception approaches to halt carcinogenesis. Epigenetic modulation by EZH2 of immune-related genes has been implicated in many cancers including colorectal but it has yet to be explored in LS carcinogenesis. The main objective of this study is to test the effect of EZH2 inhibition on immune cell profiling, colorectal carcinogenesis, gene expression, and histone methylation, using ex vivo organoids and a LS mouse model. Methods: A mouse model of LS colorectal carcinogenesis (Villin-Cre;Msh2fl/fl;TgfβRII-KI; herein referred to as VCMsh2THu) and ex vivo colonic organoids were utilized to assess the effects of the EZH2 inhibitor GSK503. Mice were treated for 9-weeks with GSK503 with downstream studies exploring polyp multiplicity, changes to the immune landscape, and epigenetic analyses to derive mechanistic insights into EZH2 regulation in LS. Results: GSK503 significantly altered transcriptomic changes in VCMsh2THu organoids with tissue-specific tropism for dysplastic organoids as compared to organoids prepared from adjacent normal mucosa. GSK503 significantly increased CD4+and CD8+ T cells in splenocytes and colonic mucosa of treated mice compared to controls in vitro and in vivo. A preventive-dose (200μg/kg) of GSK503 for 9 weeks significantly reduced adenoma multiplicity in the large intestine of VCMsh2THu mice with significant changes in the immune landscape of treated mice compared to controls. The data suggests GSK503 significantly enriched both total and activated cytotoxic T-lymphocytes CD8+/CD137+), stromal macrophages (CD68+), and activated helper T-lymphocytes (CD4+/CD134+) in the large intestine of VCMsh2THu mice. Transcriptome and molecular analyses using IHC, Western blots, and computational tools showed activation of immune and apoptotic markers with concomitant reduction of H3K27 methylation levels in colonic crypts. ChIPseq analyses revealed decreased levels of H3K27me3 and H3K4me1 (enhancer), a slight increased levels of H3K27ac (enhancer) marks with no changes in levels of H3K4me3 in treated mice, indicating that EZH2 inhibition yields global reprograming of poised promoters and active enhancers in the LS mouse genome. Conclusions: Collectively, our findings reveal that GSK503 has a promising potential as an immuno-interception modality establishing the framework for launching phase I clinical trials with EZH2 inhibitors for LS carriers. Citation Format: Fahriye Duzagac, Charles M. Bowen, Laura Reyes-Uribe, Mahira Zaheer, Jacklyn Thompson, Nan Deng, Melissa Taggart, Abhinav Jain, Winfried Edelmann, Krishna M. Sinha, Eduardo Vilar. Inhibition of histone methyltransferase EZH2 for immune-interception of colorectal cancer in Lynch syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7552.

Abstract 1540: The dendritic cell receptor DNGR-1 shapes immunity to cancer

Abstract Background: Central to the success of CD8+ T cell-mediated anti-cancer response are type 1 conventional dendritic cells (cDC1s), acting as professional antigen presenting cells. In both mouse and human, DNGR-1 (a.k.a. CLEC9A) is a dedicated cDC1 receptor that, upon binding to its physiological ligand filamentous-actin (F-actin) exposed upon cell death, prime cytotoxic CD8+ T cells specifically against dead cell-associated antigens. Here, we interrogate the role of DNGR-1 in shaping the tumor microenvironment during carcinogenesis. Methods: We utilized two models of chemical carcinogenesis: 3-methylcholanthrene (MCA)-induced sarcoma, and azoxymethane (AOM) and dextran sodium sulfate colitis (DSS)-associated carcinoma. Rag1−/- (no B and T cells), Clec9agfp/gfp, Clec9acre/cre and WT (wildtype) mice on C57BL/6 background were used. Primary MCA-sarcoma cell lines were generated and subsequently tested in a series of transplantation experiments in naïve syngeneic WT mice. Whole exome and RNA sequencing were performed to elucidate the mutational landscape and decipher the immunopeptidome of these primary cell lines. Results: Upon challenge with MCA, there is shorter latency for tumorigenesis in DNGR-1-deficient mice (n=46), like in Rag1−/− mice (n=43), in contrast to immunocompetent WT hosts (n=59) (median: 95 vs 113 days, hazard ratio: 1.72, 95% CI 1.11-2.68, p=0.008). DNGR-1-deficient mice also display increased susceptibility to AOM-DSS carcinogenesis compared to co-housed WT mice. Majority (n=16/24, 68%) of MCA-induced primary sarcoma cell lines derived from DNGR-1-deficient hosts are highly immunogenic and were controlled/rejected at a similar rate, upon challenge in secondary naïve WT recipients, like those derived from Rag1−/− mice (n=13/19, 68%). Whole-exome sequencing of cell lines derived from DNGR-1-deficient hosts revealed that they harbor high mutational burden, including mutations in F-actin-binding proteins that have been previously associated with better survival outcomes in patients with cancer (Giampazolias et al. Cell, 2021). Collectively, these data argue that DNGR-1 protects against carcinogenesis and selects for less immunogenic tumor cells. To evaluate the degree of immunoediting which occurred in the respective primary host mice, we applied the evolutionary metric based on the ratio of nonsynonymous to synonymous mutations (dN/dS), and found that they are less immunoedited compared to cell lines derived from WT hosts. Conclusion: Our findings highlight DNGR-1 as a novel immune checkpoint that couples cell death sensing and (neo-)antigen cross-presentation to functional tumor-specific CD8+ T cell responses during cancer immunoediting. Understanding the mechanism by which DNGR-1 sculpts the mutational landscape of tumors may further inform the dynamics of cancer evolution and may have translational therapeutic implications in directing effective immunotherapy strategies. Citation Format: Kok Haw Lim, Evangelos Giampazolias, Bruno Frederico, Neil C. Rogers, Probir Chakravarty, Gavin Kelly, Ana Cardoso, Emma Nye, Alejandro Suárez-Bonnet, Simon Priestnall, Robert Goldstone, Cell Services Science Technology Platform, Sonia Lee, Oliver Schulz, Jessica Strid, Luis Zapata, Caetano Reis e Sousa. The dendritic cell receptor DNGR-1 shapes immunity to cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1540.

Abstract 1471: A novel mouse model of upper tract urothelial carcinoma influenced by gut microbiota

Abstract Introduction & Objective: Recently, it has been revealed that bladder cancer (BC) and upper tract urothelial carcinoma (UTUC) are biologically distinct diseases. Mouse models of BC carcinogenesis have been established using N-butyl-N-(4-hydro-oxybutyl) nitorosamine (BBN) for a long time, but there are few reports of UTUC carcinogenesis with high incidence. Therefore, the UTUC carcinogenesis mouse model is expected. The aim of this study is to establish a novel mouse model of UTUC carcinogenesis and to identify the characteristics of this model. Methods: We used two strains (C57BL/6 and BALB/c) of male and female mice and fed 0.05% BBN from 6 weeks old to the harvest day. Both sides of upper urinary tract from mice drinking BBN or tap water were subjected to whole exome sequencing and whole transcriptome sequencing (WTS) to assess its genetic similarity to human UTUC. Microbiome in gut were assessed by 16S ribosomal RNA gene sequencing (16S rRNA-seq). Metabolic differences in feces, serum and liver between UTUC mice and healthy mice were evaluated by comprehensive metabolomic analysis. Inflammatory mediators were assessed by quantitative polymerase chain reaction (qPCR). Results: Only BALB/c female showed higher incidence of UTUC than BC, while the other strains rarely had UTUC. This model showed SBS5-like mutational pattern. The frequent mutated gene list showed similarity to human UTUC. In terms of gene expression, this model belonged basal subtype. We detected 593 differentially expressed genes between cancerous side and non-cancerous side of upper urinary tract in this model, which could also classify human UTUC and adjacent normal samples. The cancerous side of upper urinary tract enriched several inflammation related pathways such as Nfkb and Tnf pathway. We validated the upregulation of inflammatory gene expression in upper urinary tract (Il6, Il1b, Tnfa, Relb) by qPCR (all: p < 0.05). 16S rRNA-seq revealed that Firmicutes was enriched in feces. Metabolomic analysis revealed that some amino acids in feces and FAD in serum were upregulated. Feeding with amino acid mediated diet changed gut environment. Firmicutes in feces and FAD in both feces and serum were downregulated consistently. This diet suppressed inflammatory gene expression in upper urinary tract (Il6, Il1b, Tnfa, Relb, Cd38, Tnfrsf1b and Kdm1a; all: p < 0.05). Finally, UTUC was disappeared but not BC in this model treated by BBN with amino acid mediated diet. Conclusion: We have established a novel mouse model developing UTUC with high incidence that genetically mimics human UTUC. In addition, amino acid mediated diet can change both microbiome and metabolome in gut of this model, downregulate host’s inflammatory reaction to BBN in upper urinary trat and suppress the UTUC carcinogenesis. Citation Format: Akinaru Yamamoto, Atsunari Kawashima, Kentaro Jingushi, Sassi Nesrine, Yuki Horibe, Akihiro Yoshimura, Masaru Tani, Liu Yutong, toshiki Oka, Yohei Okuda, toshihiro Uemura, Gaku Yamamichi, Yu Ishizuya, Yoshiyuki Yamamoto, Taigo Kato, Koji Hatano, Norio Nonomura. A novel mouse model of upper tract urothelial carcinoma influenced by gut microbiota [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1471.

The immunomodulatory impact of naturally derived neem leaf glycoprotein on the initiation progression model of 4NQO induced murine oral carcinogenesis: a preclinical study.

Murine tumor growth restriction by neem leaf glycoprotein (NLGP) was established in various transplanted models of murine sarcoma, melanoma and carcinoma. However, the role of NLGP in the sequential carcinogenic steps has not been explored. Thus, tongue carcinogenesis in Swiss mice was induced by 4-nitroquinoline-1-oxide (4NQO), which has close resemblance to human carcinogenesis process. Interventional role of NLGP in initiation-promotion protocol established during 4NQO mediated tongue carcinogenesis in relation to systemic immune alteration and epithelial-mesenchymal transition (EMT) is investigated. 4NQO was painted on tongue of Swiss mice every third day at a dose of 25µl of 5mg/ml stock solution. After five consecutive treatment with 4NQO (starting Day7), one group of mice was treated with NLGP (s.c., 25µg/mice/week), keeping a group as PBS control. Mice were sacrificed in different time-intervals to harvest tongues and studied using histology, immunohistochemistry, flow-cytometry and RT-PCR on different immune cells and EMT markers (e-cadherin, vimentin) to elucidate their phenotypic and secretory status. Local administration of 4NQO for consecutive 300 days promotes significant alteration in tongue mucosa including erosion in papillae and migration of malignant epithelial cells to the underlying connective tissue stroma with the formation of cell nests (exophytic-hyperkeratosis with mild dysplasia). Therapeutic NLGP treatment delayed pre-neoplastic changes promoting normalization of mucosa by maintaining normal structure. Flow-cytometric evidences suggest that NLGP treatment upregulated CD8+, IFNγ+, granzyme B+, CD11c+ cells in comparison to 4NQO treated mice with a decrease in Ki67+ and CD4+FoxP3+ cells in NLGP treated cohort. RT-PCR demonstrated a marked reduction of MMP9, IL-6, IL-2, CD31 and an upregulation in CCR5 in tongues from 4NQO+NLGP treated mice in comparison to 4NQO treated group. Moreover, 4NQO mediated changes were associated with reduction of e-cadherin and simultaneous up-regulation of vimentin expression in epithelium that was partially reversed by NLGP. Efficacy of NLGP was tested first time in sequential carcinogenesis model and proved effective in delaying the initial progression. NLGP normalizes type 1 immunity including activation of the CD8+T effector functions, reduction of regulatory T cell functions, along with changes in EMT to make the host systemically alert to combat the carcinogenic threat.