Pirc Rats Research Articles

Gut microbiota drives colon cancer risk associated with diet: a comparative analysis of meat-based and pesco-vegetarian diets

BackgroundColorectal cancer (CRC) risk is strongly affected by dietary habits with red and processed meat increasing risk, and foods rich in dietary fibres considered protective. Dietary habits also shape gut microbiota, but the role of the combination between diet, the gut microbiota, and the metabolite profile on CRC risk is still missing an unequivocal characterisation.MethodsTo investigate how gut microbiota affects diet-associated CRC risk, we fed Apc-mutated PIRC rats and azoxymethane (AOM)-induced rats the following diets: a high-risk red/processed meat-based diet (MBD), a normalised risk diet (MBD with α-tocopherol, MBDT), a low-risk pesco-vegetarian diet (PVD), and control diet. We then conducted faecal microbiota transplantation (FMT) from PIRC rats to germ-free rats treated with AOM and fed a standard diet for 3 months. We analysed multiple tumour markers and assessed the variations in the faecal microbiota using 16S rRNA gene sequencing together with targeted- and untargeted-metabolomics analyses.ResultsIn both animal models, the PVD group exhibited significantly lower colon tumorigenesis than the MBD ones, consistent with various CRC biomarkers. Faecal microbiota and its metabolites also revealed significant diet-dependent profiles. Intriguingly, when faeces from PIRC rats fed these diets were transplanted into germ-free rats, those transplanted with MBD faeces developed a higher number of preneoplastic lesions together with distinctive diet-related bacterial and metabolic profiles. PVD determines a selection of nine taxonomic markers mainly belonging to Lachnospiraceae and Prevotellaceae families exclusively associated with at least two different animal models, and within these, four taxonomic markers were shared across all the three animal models. An inverse correlation between nonconjugated bile acids and bacterial genera mainly belonging to the Lachnospiraceae and Prevotellaceae families (representative of the PVD group) was present, suggesting a potential mechanism of action for the protective effect of these genera against CRC.ConclusionsThese results highlight the protective effects of PVD while reaffirming the carcinogenic properties of MBD diets. In germ-free rats, FMT induced changes reminiscent of dietary effects, including heightened preneoplastic lesions in MBD rats and the transmission of specific diet-related bacterial and metabolic profiles. Importantly, to the best of our knowledge, this is the first study showing that diet-associated cancer risk can be transferred with faeces, establishing gut microbiota as a determinant of diet-associated CRC risk. Therefore, this study marks the pioneering demonstration of faecal transfer as a means of conveying diet-related cancer risk, firmly establishing the gut microbiota as a pivotal factor in diet-associated CRC susceptibility.6mA3tL1nA724D7oE9_suJhVideo

Abstract 762: The combination of EPA and naproxen suppresses colon tumorigenesis via altering the TME, and promoting epithelial maturation in cancerous crypts

Abstract Introduction: Colorectal cancer (CRC) is a leading cause of cancer-associated mortality. Our recent study in Pirc rats revealed that the combination of eicosapentaenoic acid and sodium naproxen exhibited additive tumor protection. While the results from this preclinical study were impressive, showing tumor suppression in excess of 95%, potential mechanisms underlying this tumor protection remain unclear. The purpose of this study was to determine the transcriptomic changes resulting in the tumor suppression afforded by the combination of EPA and naproxen supplementation in the diet. Methods: Six week-old Pirc rats were fed either a control AIN-93G diet, or AIN-93G diet supplemented with 2% EPA combined with 200 ppm naproxen, for a total of 20 weeks. Global transcriptomic analysis was performed using RNA sequencing (RNA-Seq) of colon tumors and tumor-adjacent normal tissue from 26-week-old Pirc rats. 100-bp paired-end sequencing was performed on an Illumina NovaSeq to a depth of 25 million reads per sample. Reads were trimmed with Trimmomatic-v0.39, and reads were mapped to the mRatBN7.2.105 rat reference genome using HISAT2. Differentially expressed genes were calculated using DESeq2, and gene set enrichment analysis (GSEA) were performed using Rstudio. Results: DESeq2 identified 809 differentially expressed genes (DEGs, 2-fold change > 1, FDR < 0.05) between large tumors from AIN-93G fed rats versus suppressed tumors from rats fed the combination of EPA and naproxen (490 up-regulated, 319 down-regulated). GSEA analysis revealed activation of inflammatory pathways including IL-17 signaling, JAK-STAT signaling, complement activation, cytokine signaling, ribosome biogenesis, and cell cycle were enriched in untreated control. In contrast, suppressed tumors from animals supplemented with EPA and naproxen were enriched in pathways including negative regulation of Wnt signaling, cell fate specification, ion channel complexes, and nutrient absorption. Furthermore, suppressed tumors shared significant GSEA pathway overlap with normal tissue, leading us to believe the combination of EPA and naproxen may be preventing tumorigenesis in part by maintaining cells in a differentiated state. Conclusion and Future directions: Inflammation plays a key role in tumorigenesis and promoting cancer stem cell niches in tumors. According to our analysis, the combination of EPA and naproxen attenuated cancer-associated inflammation and reduced proliferation in part by augmenting tumor differentiation. Future studies will utilize single-nuclei RNA-Seq (snRNA-Seq) to determine at a single cell resolution the transcriptomic changes of epithelial and stromal populations within the TME. This work was supported by National Cancer Institute task order 75N91019F00132. Citation Format: Ryan Beach, Michael Martinez, Vignesh Gunasekharan, Daniel W. Rosenberg. The combination of EPA and naproxen suppresses colon tumorigenesis via altering the TME, and promoting epithelial maturation in cancerous crypts [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 762.

Abstract 5276: Immunomodulatory activity of intermittent low-dose erlotinib plus sulindac: Implications for hereditary and sporadic colorectal cancer

Abstract A once-weekly optimized dosing regimen of erlotinib (ERL) in the polyposis in rat colon (Pirc) model [1] was a reliable predictor of antitumor efficacy in familial adenomatous polyposis (FAP) patients [2]. Pirc rats are increasingly used as a translational tool for FAP studies. Based on transcriptomic data from FAP patients receiving ERL plus sulindac (SUL) vs. placebo [3], we determined the timing of changes in interferon-γ signaling in Pirc adenomas, while performing additional dose titration. In rats that were given ERL administered at 5 mg per kilogram of body weight through oral gavage (two times a week) and that received a diet containing 125 parts per million of SUL, a regimen referred to as SUL125+ERL5x2, significant suppression of tumor growth was observed. This combination represented half of the standard of care SUL dose and about one-quarter of the ERL dose in FAP patients. In adenomas collected after one year of treatment from SUL125+ERL5x2 treated rats, the expression of genes related to the major histocompatibility complex (MHC) class I (β2m, Cnx, Psmb8 and Tap1) and MHC class II (RT1-A2, Cd74, RT-1Bb, Cd74, and Ciita) was increased, compared to vehicle controls, excluding a subset of adenomas that remained resistant. Through the use of multiplex mass cytometry (CyTOF), an increase in tumor-infiltrating CD4+ T cells was observed in adenomas from the SUL125+ERL5x2 treatment group. Administration of single or combined agents increased CD68+ cells and reduced the presence of Foxp3+ and Arg1+ cells in Pirc adenomas. The natural history of adenomatous colon polyps indicated an increase in MHC gene expression as tumors increased in size; however, this trend reversed markedly in fully occluding lesions. Additionally, expression of MHC-related factors increased in Pirc colon adenoma and murine colon carcinoma cells treated with ERL±SUL, resulting in enhanced CD8+ T-cell activation and IL-2 secretion. In conclusion, treatment with intermittent low doses of ERL±SUL increased MHC gene expression and induced changes in the immune cell component in the tumor microenvironment of Pirc rat adenomas. A subset of non-responsive adenomas could benefit from additional immunopreventive approaches, with implications for possible clinical applications in both hereditary and sporadic colorectal cancer.

Abstract 7299: Preventing CRC progression in FAP rat model using a combinational targeting of TRAIL signaling and inflammation

Abstract Colorectal Cancer (CRC) is among the leading causes of mortality globally, with high incidence rates in developed countries including the US. While FAP patients are prone to develop CRC in their lifetime, recent rising trends among young adults is also a major concern. Hence, preventing CRC would aid in reducing cancer mortality, especially in high-risk FAP patients for which there are no FDA approved preventive agents. Here, we evaluated TRAIL-inducing, orally active small molecule ONC201, alone and in combination with the NSAID naproxen (NAP) for CRC prevention in the PIRC rat model representing FAP cohort. Male PIRC rats were bred inhouse and randomized by age into placebo and intervention groups (N=15). Starting at 6 weeks of age, experimental groups were administered either ONC201 alone by gavage [25mg/kg; 2X/week or 50mg/kg; 1X/week]; NAP in AIN-76A diet [200 ppm; continuously or 1 week ON/OFF]; or a combination of both agents, while placebo group received vehicle only. After 26 weeks of treatment, rats were euthanized and intestines were evaluated for tumor incidence and multiplicity. H&E stained colonic tumor sections were histologically classified as hyperplastic polyps, adenomas (AD), and adenocarcinomas (ADCA). Gross tumor data indicated significant reduction in colonic polyps in both the NAP alone and NAP+ONC201 groups without any overt-toxicites. Histological analyses also demonstrated strong suppression of AD progression to ADCA as well as a significant decrease in the number of ADs and ADCAs (p<0.05) in the treatment groups compared to the placebo. ONC201 regimens modestly reduced AD (19%-27% less, p<0.05) but showed strong inhibition of ADCA (45%-59% less, p<0.0001). A significant reduction of AD and ADCA was seen in NAP continuous (54% & 87% less respectively; p<0.0001) and intermittent (34% & 68% less respectively; p<0.0001) dosing regimens. As anticipated, the NAP continuous treatment had better efficacy than the intermittent dosing regimen. Notably, the combination treatment showed >90% (p<0.0001) inhibition of ADCA and 40%-57% (p<0.0001) inhibition of ADs. Importantly, ONC201+ NAP treatment also resulted in an additional 45% - 70% reduction of ADCA (p<0.0001) when compared to the NAP alone treatment. Thus, our data showed that the combination groups with NAP intermittent dosing demonstrated ~90% inhibition of ADCA, suggesting the synergistic effect of the two agents in preventing CRC progression. Molecular analysis of the rat tumors and additional analyses in in-vitro tumoroid models suggested induction of TRAIL (TRAIL, DR5, FADD), increased apoptosis (Caspases), and a decrease in proliferation markers (PCNA, Cyclin D1) and pro-inflammatory cytokines (IL1b, IL13 etc.). Our data suggests NAP/ONC201 combo has no toxicities, warranting further investigation of CRC interception in high-risk FAP patients (Funding by NCI-PREVENT 75N91019D00020-75N91020F00004). Citation Format: Venkateshwar Madka, Gopal Pathuri, Karthikkumar Venkatachalam, Surya P. Singh, Anil Singh, Nicole Stratton, Stanley Lightfoot, Shizuko Sei, Mark S. Miller, Chinthalapally V. Rao. Preventing CRC progression in FAP rat model using a combinational targeting of TRAIL signaling and inflammation [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 7299.

Abstract LB114: Colon cancer preventive efficacy of ONC201 and naproxen alone or in combination in FAP relevant PIRC rat model

Abstract Colorectal Cancer (CRC) is the third most common cancer and leading cause of mortality in the US. While the rate of CRC incidence has steadily dropped in the last decades, recent rising trends among young adults are a major concern. Hence, preventing CRC in the general population would aid in reducing cancer mortality, especially in high-risk FAP patients for which there are no FDA approved preventive agents. Here, we evaluated TRAIL-inducing agent ONC201, alone and in combination with NSAID naproxen (NAP) for CRC prevention in PIRC rat model of FAP. PIRC rats (20 female rats/group) are bred and randomized by age into control and intervention groups. Starting at 6 weeks of age, PIRC rats were treated with ONC201 by gavage [25mg/kg; 2X/week (LD) or 50mg/kg; 1X/week (HD)]; NAP 200 ppm in AIN-76A diet [continuously or 1 week ON/OFF]; or the combination of ONC201 and NAP. Control group rats received vehicle only. Colonic polyps’ (CP) development (occurrence, number, and size) was monitored by periodic colonoscopies and upon termination at 32 weeks of age. Colonoscopy data indicated significantly fewer CPs in the HD ONC201, NAP alone dosing regimens, and combination groups. At termination, colonic tumors were harvested and subjected to histopathological evaluation. Colonic tumors were histologically classified as hyperplastic polyps, adenomas, and adenocarcinomas (ADCA). In vehicle treated rats, histological assessment showed multiplicity of 2.4±0.4 (Mean±SEM) hyperplastic polyps; 9.14±0.92 adenomas, and 2.4±0.27 ADCA. A significant decrease in the number of hyperplastic polyps (p<0.05) was observed in rats treated with combination of ONC201 and NAP continuous dosing regimens. LD and HD ONC201 regimens resulted in modest (11.3%, p=0.45) and significant (48.8%, p<0.0001) inhibition of colonic adenoma multiplicity, respectively. As anticipated, NAP dosing regimens showed 78-85% inhibition (p<0.0001) of adenoma multiplicity. Combination treatment showed >90% inhibition of adenomas. Importantly, LD ONC201 inhibited colonic ADAC multiplicity by 78.8% (p<0.0001) and remaining treatment groups showed no colonic ADACs. This histological data clearly showed that ONC201 (25 mg 2X/week or 50 mg 1x/week) significantly suppresses colonic tumor progression to colon adenoma and adenocarcinomas. NAP, either continuously or intermittent dosing, resulted in 85% and 78% inhibition of adenoma multiplicities, respectively, suggesting that intermittent dosing may be similarly effective for CRC prevention. Biomarker analysis suggested increased apoptosis (TRAIL, Caspase-3) and decrease in proliferation markers (PCNA, Cyclin D1). Our data warrants investigating ONC201 with NAP combination for CRC prevention in high risk FAP patients. (Supported by NCI-PREVENT 75N91019D00020-75N91020F00004) Citation Format: Venkateshwar Madka, Gopal Pathuri, Karthikkumar Venkatachalam, Srikanth Chiliveru, Yuting Zhang, Nandini Kumar, Nicole Stratton, Stanley Lightfoot, Mark S. Miller, Shizuko Sei, Chinthalapally V. Rao. Colon cancer preventive efficacy of ONC201 and naproxen alone or in combination in FAP relevant PIRC rat model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB114.

Combination of naproxen and a chemically-stable eicosapentaenoic acid analog provide additive tumor protection in Pirc rats.

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the United States. Patients with the genetic disorder Familial Adenomatous Polyposis (FAP) develop hundreds to thousands of polyps that unless removed by prophylactic colectomy will progress to CRC at an early age. Nonsteroidal anti-inflammatory drugs (NSAIDs) and the ω-3 polyunsaturated fatty acid (PUFA) eicosapentaenoic acid (EPA), have been evaluated for their chemopreventive potential in delaying CRC onset in high-risk patients. In our study, we determined whether the NSAID, naproxen, alone or in combination with a chemically-stable EPA analog (TP-252), affects tumor formation in the ApcPirc rat model. When compared to control diet, animals fed naproxen or HD TP-252 had 66% and 82% fewer tumors, respectively. However, animals fed a combination of naproxen and HD TP-252, exhibited a 95% reduction in tumor formation and a 98% reduction in tumor volume, respectively. To elucidate potential mechanisms of tumor protection, a comprehensive, targeted lipidomic analysis was performed on colonic mucosa to determine changes in eicosanoid metabolism. Animals receiving TP-252 alone or in combination with naproxen had significantly reduced mucosal levels of proinflammatory ω-6 eicosanoids (PGE2 , 5-HETE and 14,15-DiHETrE), along with a simultaneous increase in anti-inflammatory EPA-derived ω-3 eicosanoids. A comprehensive lipidomic analysis also uncovered several potential pharmacodynamic (PD) lipid biomarkers, including resolvin E2, 9-HEPE, 12-HEPE and 18-HEPE, that were significantly correlated with tumor protection. Further studies with this drug combination should be focused on dose optimization and the role of EPA-derived lipid mediators in CRC initiation and progression.

Abstract A005: Identification of lipid pharmacodynamic biomarkers in FAP model

Abstract Familial Adenomatous Polyposis (FAP) is a genetic disorder characterized by the development of numerous polyps in the colon, that unless removed by colectomy, inevitably progress to colorectal cancer (CRC). Although there is currently no FDA-approved treatment to delay colectomy, a common target for chemoprevention is cyclooxygenase-2 (COX-2). Non-steroidal anti-inflammatory drugs (NSAIDs) and omega-3 fatty acids are known to alter COX metabolism and affect downstream eicosanoids involved in CRC progression. In this study, we examined the chemopreventive efficacy of the NSAID naproxen alone, or in combination with TP-252, a novel, chemically-stable analogue of the omega-3 fatty acid, eicosapentaenoic acid (EPA), in the intestinal tumorigenesis Apcam1137 PIRC rat model. Over the course of this 20-week study, rats were fed a modified AIN-93G diet supplemented with 200 ppm naproxen, alone or in combination with TP-252 (1.5%, or 3% by weight). Rats treated with 200 ppm naproxen had 66% (p < 0.0001) fewer colon tumors than controls (AIN-93G) and an 81% (p < 0.0001) reduction in tumor size. However, when Naproxen was combined with high-dose TP-252 (3%), tumor number and size were reduced by 95% (p < 0.0001) and 98% (p < 0.0001) respectively, when compared to controls. Since the primary mechanisms of action for naproxen and EPA target COX metabolic activity, we performed a comprehensive lipidomic analysis on colon tissue and plasma to determine lipidomic changes associated with drug treatment. Increased formation of several EPA-derived eicosanoids, including 9-HEPE, 12-HEPE, 18-HEPE, and resolvin E2, whose mucosal levels correlated with both reduced tumor number and volume, were observed. These metabolites were also concomitantly elevated in the plasma of treated animals, representing potential pharmacodynamic (PD) biomarkers of CRC prevention. Future studies will decipher potential mechanisms that underlie these EPA-derived PD biomarkers and their role in tumor suppression. (Supported by the NCI Prevent Program 75N91019D00019, Task Order 75N91019F00132). Citation Format: Ryan Beach, Altaf Mohammed, Daniel W. Rosenberg. Identification of lipid pharmacodynamic biomarkers in FAP model [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr A005.

Abstract IA005: Discovery and development of LFA-9 for CRC prevention

Abstract Colorectal Cancer (CRC) is a major public health issue with an estimated 980,000 deaths annually, worldwide. Current trends show an increase of CRC incidence and mortality worldwide. Colonic adenomas are very common in ages 50 and over. Though, nonsteroidal antiinflammatory agents like Aspirin, Naproxen and COX-2 inhibitor and Celecoxib are useful to prevent the polyp progression and reducing CRC burden, continuous/chronic usage of these drugs are limited by GI toxicity and unwanted side effects. Thus, the rationale to establish safer anti-inflammatory agents for CRC prevention is important. Mechanistic studies suggest that up-regulation of 15-PGDH, sparing COX-1/2 and prostaglandin (PG)I2 synthase and/or selectively targeting mPGES-1 and 5-LOX would reduce the cardiovascular side effects and may improve the chemopreventive efficacy. There are limited to no studies on targeting mPGES-1/5LOX and it is one of the unexplored areas in colorectal cancer chemoprevention towards safer drug development. To design and develop selective inhibitors of dual mPGES-1/5-LOX, we used in-silico small molecular docking simulation approaches, and identified LFA-9 as a novel duel mPGES-1/5-LOX inhibitor among >35 analogs. Azoxymethane (AOM)-induced rat colonic tumors were utilized as ex-vivo to assess pharmacodynamic inhibitory effects of LFA-9 on mPGES-1 and 5-LOX by Radio-HPLC. In a series of animal experiments, we established the dose-range of toxicity and optimal doses of LFA-9. Potential preventive efficacy of LFA-9 was assessed AOM-rat colon carcinogenesis (Aberrant Crypt Foci, (ACF) as surrogate marker in male F344 rats and intestinal tumor inhibition in APCMin/+ mice. Furthermore, we have shown the efficacy of LFA-9 administered in the diet at the adenoma stage significantly suppressed colonic adenocarcinoma formation in AOM-induced colon cancer (sporadic CRC model) and PIRC rat model (FAP model). Overall, the above results show that LFA-9, a novel dual mPGES-1/5-LOX inhibitor, is a safer agent and has potential for prevention of CRC in high-risk patients. {This work was supported by NCI-PREVENT Cancer Program, R01 CA213987 and VA Merit Awards} Citation Format: Chinthalapally V. Rao. Discovery and development of LFA-9 for CRC prevention [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr IA005.

Comparison of Absolute Expression and Turnover Number of COX-1 and COX-2 in Human and Rodent Cells and Tissues.

ObjectiveWe aim to quantify the absolute protein expression of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) in various cells and tissues to determine the relative contribution of COX-1 and COX-2 to PGE2 production.MethodsAn LC-MS method was developed and validated, then used for quantifying the absolute amounts of COX-1 and COX-2 in recombinant human COX-1 and COX-2, lysates from different cells, tissue microsomes of rodents and humans, Pirc rat colonic polyps, and biopsy specimens from squamous cell carcinoma (SCC) patients. The COX-1 and COX-2 turnover numbers were subsequently calculated based on apparent formation rates of PGE2.ResultsA robust LC-MS method for quantification of COX-1 and COX-2 was developed and validated and then used to calculate their apparent turnover numbers. The results showed that COX-1 expression levels were much higher than that of COX-2 in all the tested tissues including the colonic epithelium of F344 (28-fold) and Pirc rats (20-fold), colonic polyps of Pirc rats (8-fold), and biopsy specimens of SCC patients (11–17-fold). In addition, both COX-1 and COX-2 were higher in polyps when compared to adjacent mucosa of Pirc rats. The turnover number of recombinant human COX-2 was 14-fold higher than that of recombinant human COX-1. LPS stimulation increased COX-2 protein expression in three cell lines (Raw 264.7, SCC9 and EOMA) as expected but unexpectedly increased COX-1 protein expression (13.8-fold) in EOMA cells.ConclusionIn human oral cancer tissues and cells as well as Pirc rat colon, COX-1 plays an unexpectedly but more important role than COX-2 in abnormal PGE2 production since COX-1 expression is much higher than COX-2. In addition, COX-1 expression levels are inducible in cells, and higher in polyps than surrounding mucosa in Pirc rat colon. These results indicate that targeted suppression of local COX-1 should be considered to reduce colon-specific PGE2-mediated inflammation.

Abstract 5631: Immunomodulatory activity of sulindac plus erlotinib for colon cancer prevention in familial adenomatous polyposis

Abstract Familial adenomatous polyposis (FAP) is an autosomal dominant disorder caused by germline mutation in Adenomatous Polyposis Coli (APC)1. Patients with FAP have a nearly 100% lifetime risk of colorectal cancer (CRC) due to the formation of hundreds to thousands of adenomatous polyps in the gastrointestinal tract2. A clinical trial in FAP patients demonstrated that Cox-1/2 inhibitor sulindac (SUL) plus EGFR inhibitor erlotinib (ERL) reduced polyp burden significantly in the colon and small intestine (SI), but toxicity issues raised concerns for durable long-term CRC interception3. In the Apc-mutant polyposis in rat colon (Pirc) rat model of FAP, we determined low-dose regimens of ERL+SUL that were both safe and efficacious4. In the current investigation, the lowest tested dose of dietary SUL (125 ppm continuously) plus ERL (5 mg/kg twice weekly), given at one-half the SUL clinical dose and one-fourth of the ERL clinical dose, administered for 46 weeks respectively, demonstrated good inhibition of tumor burden in the colon (>85% efficacy, p<0.05) and SI (>94% efficacy, p<0.05) compared to untreated controls. Moreover, treatment normalized organ weights and hematocrits that were altered in the untreated AIN controls.Recently, ERL5 and SUL6 were individually demonstrated to increase the recruitment of inflammatory immune cells in mouse models of lung and colon cancer, respectively. However, ERL+SUL in combination was not investigated in the context of immune cell infiltration and polarization. Our preliminary IHC data in the Pirc model indicated increased recruitment of CD8+ T cells in colon polyps by SUL 250 ppm + ERL 42 mg/kg once a week. RT-qPCR and immunoblotting analysis of colon polyps obtained from the low-dose combination of SUL 125 ppm + ERL 5 mg/kg twice weekly demonstrated increased CD4 expression and concurrently decreased oncogenic IL-17 expression and tumor associated macrophage markers (CD163 and CD206) (P<0.05 vs control). Additional immune markers will be identified and validated via CyTOF. We hypothesize that low-dose combination of SUL+ERL can enhance the recruitment of immune cells into early adenomatous colon and SI polyps, providing an effective immunointerception strategy, with potential clinical relevance for FAP patients before or after colectomy. Acknowledgements Research supported by NCI Contract Number 75N91019D00021, Task Order 75N91019F00130. 1.Groden J et al., Cell 1991; 66:589-600.2.Jasperson KW et al., Gastroenterology 2010; 138:2044-2058.3.Samadder NJ et. al., JAMA 2016; 315:1266-75.4.Ulusan AM et. al., Cancer Prev Res (Phila). 2021; 14:325-336.5.Ayeni D et al., J Immunother Cancer 2019; 7:172.6.Yi B et al., Mol Cancer Ther 2021; Apr 20. Citation Format: Chakrapani Tripathi, Roderick H. Dashwood. Immunomodulatory activity of sulindac plus erlotinib for colon cancer prevention in familial adenomatous polyposis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5631.

Intestinal microbiota profiles in a genetic model of colon tumorigenesis correlates with colon cancer biomarkers

Faecal (FM) and colon mucosal associated microbiota (MAM) were studied in a model of colorectal cancer (CRC), the Apc-mutated Pirc rats, and in age-paired wt F344 rats. Principal Coordinates Analysis indicated that samples’ distribution was driven by age, with samples of young rats (1 month old; without tumours) separated from older ones (11-month-old; bearing tumours). Diversity analysis showed significant differences between FM and MAM in older Pirc rats, and between MAM of both Pirc and wt rats and the tumour microbiota, enriched in Enterococcus, Escherichia/Shigella, Proteus and Bifidobacteriaceae. In young animals, Pirc FM was enriched in the genus Delftia, while wt FM was enriched in Lactobacillus and Streptococcus. Some CRC biomarkers and faecal short chain fatty acids (SCFAs) were also measured. Colon proliferation and DClK1 expression, a pro-survival mucosal marker, were higher in Pirc than in wt rats, while the mucin MUC2, was lower in Pirc rats. Branched SCFAs were higher in Pirc than in wt animals. By Spearman analysis CRC biomarkers correlated with FM (in both young and old rats) and with MAM (in young rats), suggesting a specific relationship between the gut microbiota profile and these functional mucosal parameters deserving further investigation.

Oleuropein-Rich Leaf Extract as a Broad Inhibitor of Tumour and Macrophage iNOS in an Apc Mutant Rat Model.

Oleuropein, the major compound found in olive leaves, has been reported to exert numerous pharmacological properties, including anti-inflammatory, anti-diabetic and anti-cancer effects. The purpose of this study was to evaluate, for the first time, the effect of oleuropein-rich leaf extracts (ORLE) in already-developed colon tumours arising in Apc (adenomatous polyposis coli) mutated PIRC rats (F344/NTac-Apcam1137). Here, we were able to investigate in parallel the anti-cancer effect of ORLE, both in vivo and in vitro, and its anti-inflammatory effect on macrophages, representing a critical and abundant population in most solid tumour microenvironment. We found that in vivo ORLE treatment promoted apoptosis and attenuated iNOS activity both in colon tumours as in peritoneal macrophages of PIRC rats. We this confirmed in vitro using primary RAW264.7 cells: ORLE reduced iNOS activity in parallel with COX-2 and pro-inflammatory cytokines, such as IL-1β, IL-6 and TGF-β. These findings suggest that ORLE possess a strong anti-inflammatory activity, which could be crucial for dampening the pro-tumourigenic activity elicited by a chronic inflammatory state generated by either tumour cells or tumour-associated macrophages.

Colon fibroblasts from Pirc rats (F344/NTac-Apcam1137 ) exhibit a proliferative and inflammatory phenotype that could support early stages of colon carcinogenesis.

The role of fibroblast APC mutation in carcinogenesis is not clear. Apc +/− colon fibroblasts have been previously characterized: however, little is known about their behavior at very early‐stage of colon carcinogenesis. We cultured colon mucosa fibroblasts (PCF, Apc +/−) of Pirc rats (F344/NTac‐Apc am1137) at an early stage of tumorigenesis, in absence of preneoplastic lesions, and of age‐matched wt (WCF): DNA damage levels, inflammatory phenotype and the expression of known markers of CAFs were analyzed. The latter were also assessed by microarray analysis on colon normal mucosa of Pirc and wt animals. PCF exhibited higher proliferative rates (P < .001) and delayed replicative senescence onset (P < .05) compared to WCF, along with a lower level of oxidative DNA damage (P < .05). Furthermore, a constitutively higher expression of COX‐2 and sensitivity to inflammatory stimuli was found in PCF compared to WCF (P < .05), accompanied by higher invasive capability (P < .05) and presence of cytoplasmic chromatin foci (cytoplasmic chromatin foci, P < .05). However, they neither expressed CAFs markers (α‐SMA, IL‐6) nor responded to CAFs activating stimuli (TGF‐β). Accordingly, CAFs markers and activating stimuli resulted down‐regulated in Pirc normal mucosa compared to wt, whereas DNA damage response and tolerance pathways were overexpressed. These data show for the first time that a proliferative and inflammatory phenotype characterizes Apc +/− colon fibroblasts since very early stages of colon tumorigenesis, and indicate a role of Apc mutation in driving fibroblast phenotypic alterations that could support the establishment of a protumorigenic environment. Early pharmacological targeting of these dysfunctions might impact on tumor prevention in FAP patients.

Abstract LB225: Colon cancer preventive efficacy of licofelone and its analogue LFA-9 in PIRC rat model of FAP

Abstract Inflammation is a key hallmark of many cancers and potent target for chemoprevention. Experimental and clinical intervention studies indicate strong cancer preventive efficacy of cyclooxygenase (COX)-2 inhibitors. Their use for chemoprevention is limited due to increased cardiovascular (CV) toxicities. Selective COX-2 inhibition diverts arachidonic acid to the 5-lipoxygenase (5-LOX) pathway resulting in accumulation of prothrombotic leukotrienes while depleting antithrombotic prostaglandin (PG)I2, leading to increased risk of CV events. To overcome side effects, balanced dual inhibitors targeting COX-2/5-LOX or microsomal PGE synthase (mPGES)-1/5-LOX enzymes are being developed for chemoprevention. In this study, the clinically advanced dual COX-2/5-LOX inhibitor, licofelone, and its glycine analogue (LFA-9), the mPGES-1/5-LOX dual inhibitor, were evaluated for colon cancer preventive efficacy in the FAP relevant PIRC rat model. In preclinical dose range finding and preliminary toxicity studies in F344 rats, dietary administration of licofelone &amp;lt;500ppm and LFA9 &amp;lt;1,600ppm for 8 weeks showed no signs of toxicity based on by body weight gain, organs histopathology, blood cell counts, and serum chemistry. For efficacy evaluation, male and female PIRC rats were randomized by age (n≥15/group/gender). All rats had pre-intervention colonoscopies (8 weeks age) to determine baseline colonic polyps and were fed AIN-76A diets containing licofelone (250 ppm), LFA-9 (800ppm) or control until 32 (male) or 40 (female) weeks of age. Colonoscopies were repeated every 8 weeks for longitudinal monitoring of tumor development. At the end of the treatment period, rats were euthanized and intestines were evaluated for tumor multiplicity as an efficacy endpoint. Colonoscopy data suggested no significant difference in pretreatment colonic polyp multiplicity. However, there was a marked delay in colon tumor development in rats in the intervention group compared to control. Colonic tumor multiplicity at termination indicated that licofelone treated male rats developed 32% less tumors (21.87±1.23 (Mean±SEM); p&amp;lt;0.0001), while LFA-9 treated male rats had 31% less tumors (22.13±1.19; p&amp;lt;0.0001) compared to controls (32.25±1.37). There was no significant difference in tumor multiplicity between licofelone and LFA-9 treatment groups. In females, licofelone treatment led to 51% inhibition of colonic tumors (9.18±1.25; p&amp;lt;0.0002) while LFA-9 treatment led to 37% inhibition (11.89±0.86; p&amp;lt;0.002) compared to control (18.91±1.88). Although licofelone trended toward better inhibitory effect in females, this was not significantly different from the LFA-9 group. Colon tumor volume and small intestinal tumor multiplicity was notably inhibited in both licofelone and LFA-9 treated rats when compared to control. Our data suggest both licofelone and LFA-9 exhibit moderate and uniform chemopreventive efficacy against FAP-associated intestinal tumorigenesis in the PIRC rat model. (Supported by NCI HHSN HHSN261201500024I) Citation Format: Venkateshwar Madka, Nagendra S. Yarla, Gopal Pathuri, Yuting Zhang, Anh Bao, Nicole C. Stratton, Anil Singh, David L. McCormick, Altaf Mohammed, Shizuko Sei, Jennifer Fox, Chinthalapally V. Rao. Colon cancer preventive efficacy of licofelone and its analogue LFA-9 in PIRC rat model of FAP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB225.

LFA‐9, a Selective Inhibitor of Microsomal Prostaglandin Synthase‐1 and 5‐Lipoxygenase: Prevention of Inflammatory and Oncologic Diseases

Cyclooxygenase‐1 (COX‐1), COX‐2 and 5‐lipoxygenase (5‐LOX) as targets for inflammatory diseases and many agents were developed and extensively used in the clinic. Chronic use of drugs that target COX‐1/COX‐2 are associated with unwanted side effects including GI toxicity and cardiovascular risk. Hence, the main aim of this work is to develop mechanistic based safer agents that would target microsomal prostaglandin synthase‐1 (mPGES‐1) (a downstream enzyme of COX‐2) and 5‐LOX, while sparing COX‐1&amp;‐2 activity. LFA‐9 was designed using structure‐based drug discovery and synthesized from licofelone by adding 2‐aminoethanoic acid (Glycine) at its carboxylic end that led to targeting mPGES‐1 and 5‐LOX but not COX‐1&amp;2. In silico, in vitro and ex vivo studies on LFA‐9 were performed using human/mouse/rat mPGES‐1 and 5‐LOX to facilitate pre‐clinical experimentation and clinical translation. In the molecular docking studies, LFA‐9 showed strong binding affinity against 5‐LOX (binding energies range between −199.85 &amp; −215.3 Kcal/mol) and mPGES‐1 (binding energies range between −238.27 &amp; −257.76 Kcal/mol), but not with COX‐1&amp;2. In cell free system studies, LFA‐9 substantially inhibited mPGES‐1 (range of IC50 between 0.52 – 1.40 μM) and 5‐LOX (range of IC50 between 0.89 – 2.75 μM) but not COX‐1&amp;2 (IC50&gt;1mM) when assayed using fluorometric and colorimetric methods. Moreover, LFA‐9 substantially inhibited PGE2 (IC50 range between 0.47 – 0.78 μM) and LTB4 (IC50 range between 0.56 – 1.24 μM) production and spared PGI2 (IC50&gt;1 mM) and TXB2 (IC50&gt;1 mM) production in LPS‐stimulated human/mouse/rat macrophages and whole blood as the cell‐based system using ELISA technique. Further, circular dichroism and isothermal calorimetric based studies demonstrated that LFA‐9 strongly binds and induces changes in secondary structure of human mPGES‐1 and 5‐LOX enzymes, thereby inhibits their activities. In addition, LFA‐9 (cLogP = 4.34) obeys Lipinsky’s rule of 5 for druglikeness and shows better water solubility unlike licofelone (cLogP = 6.15). Furthermore, LFA9 prevented tumor stemness of APC mutant colonic epithelial cells from PIRC rats in vitro and inhibited exogenous PGE2 in tumor like microenvironment. Therefore, LFA9 is a novel mPGES‐1 and 5‐LOX inhibitor and safer drug candidate for inflammatory and oncologic diseases.Support or Funding InformationAuthors acknowledged NIH/NCI R01CA 213987 and NIH/NCI HHSN261201500024I for funding support.Chemical structures of Licofelone and LFA9Figure 1

DNA damage in colon mucosa of Pirc rats, an Apc-driven model of colon tumorigenesis

APC mutation is the first event triggering colon carcinogenesis (CRC). The contribution of APC to colon mucosa DNA damage is not well characterized yet. Similarly, the role of genotoxin-producer gut microorganisms is unclear. DNA strand breaks and oxidative damage were measured in Pirc rats, mutated in Apc, with the COMET assay at age 1 (T1) and 11 months (T11), i.e. in absence and presence of colon adenomas. In Pirc colon mucosa a 2-fold increase in the mean level of DNA oxidative damage was found at T11 compared to T1. Moreover, the analysis of DNA damage distribution showed that the proportion of Pirc mucosa cells in the highest DNA damage class was increased compared to wt rats at T1 and T11 months (p < 0.05 and <0.001, respectively). The analysis of colon mucosa-associated microbiota composition showed that this result was not attributable to the presence of genotoxin-producer bacteria B. fragilis nor E. coli. However, Pirc colon mucosa was enriched in Clostridium cluster XI, harmful bacteria in the large intestine, while the wt colon mucosa was enriched in Clostridium cluster IV. This work provides an original way to investigate the interplay between Apc and gut microbiota in affecting DNA stability during CRC.

Supplementation with phytoestrogens and insoluble fibers reduces intestinal carcinogenesis and restores ER-β expression in Apc-driven colorectal carcinogenesis.

Supplementation with phytoestrogens and insoluble fibers has been reported to reduce duodenal polyps in colectomized familial adenomatous polyposis patients, with a mechanism involving, at least in part, upregulation of estrogen receptor-β subtype, whose expression is lowered during intestinal tumorigenesis. These data suggest a protective effect also in the colon, the main target organ for tumorigenesis in familial adenomatous polyposis and a major cancer type in non-familial (sporadic) cancers. Therefore, we tested whether a similar preparation might reduce tumorigenesis in the colon of Pirc rats (F344/NTac-Apc) mutated in the Apc gene and thus, like familial adenomatous polyposis patients, spontaneously developing multiple tumors in the colon. We first demonstrate that estrogen receptor-β expression in Pirc rat colon is significantly down-regulated compared to age-matched wt rats. Then, Pirc rats aged 1 month were treated for 3 months with Adipol (Adi), a patented preparation containing phytoestrogens and insoluble fibers. Colon tumorigenesis was significantly reduced by Adi treatment (colon tumors/rat were 5.3 ± 0.8 and 2.9 ± 0.3, Mucin Depleted Foci/rat 127 ± 6.6 and 97.1 ± 8.6 in Controls and Adi-treated rats, respectively, means ± SE, P < 0.01). The treatment also normalized colon proliferation pattern along the crypt and significantly increased apoptosis in colon tumors. Estrogen receptor-β expression was increased by Adi treatment, especially in the tumors. These positive effects suggest that Adipol may be exploited as a chemopreventive agent to reduce cancer risk in familial adenomatous polyposis patients and to postpone prophylactic colectomy. Moreover, given the similarities between familial adenomatous polyposis and sporadic colorectal cancer, it might also be used as chemopreventive agent in colorectal cancer patients at risk.

Acute changes in colonic PGE2 levels as a biomarker of efficacy after treatment of the Pirc (F344/NTac-Apcam1137) rat with celecoxib.

This study sought to evaluate short-term treatment with COX-2 inhibitors and acute changes in colonic PGE2 levels as predictors of long-term efficacy in a genetic model of colorectal cancer. Celecoxib oral suspension (40 mg/kg BID) was dosed to Apc-mutant Pirc (F344/NTac-Apcam1137) rats for 4 days (short-term group), or the equivalent dose of 1500 ppm celecoxib was administered in the diet for 4 months (long-term group). Percent inhibition of colonic PGE2 was calculated, and the reduction in colonic PGE2 was assessed in relation to suppression of adenomatous colon polyps. Colonic mucosa PGE2 was fourfold higher in Pirc than in F344 wild-type rats (21 vs. 5.6 pg/mg epithelial tissue), due at least in part to higher COX-2 expression, and this was confirmed by elevated PGE2-d11 levels in Pirc colonic S9 incubations. In the 4-day study, dose-dependent reductions in PGE2 were observed in colonic epithelium (-33% (P>0.05) and -57% (P=0.0012)), after low- and high-dose celecoxib treatments of 4 mg/kg and 40 mg/kg (bid), respectively. In the 4-month study, 1500 ppm celecoxib suppressed colonic epithelium PGE2 by 43.5%, and tumor multiplicity by 80% (P<0.0015). Suppression of plasma 6-keto PGF1α also was corroborated following long-term treatment with 1500 ppm celecoxib (P<0.05). Acute changes in colonic mucosa PGE2 provided a rapid means of predicting long-term chemopreventive effects from celecoxib, and might be useful for screening of new COX-2 inhibitor compounds.

High Sensitivity to Cholic Acid-induced Colonic Tumorigenesis Makes Female PIRC Rats (F344/NTac-Apcam1137) a Suitable Model for Studying CRC-promoting Agents.

Rats of the adenomatous polyposis coli (Apc)-mutated female polyposis in rat (PIRC) (F344/NTac-Apcam1137) model exhibit a low level of intestinal tumorigenesis and are thus potentially exploitable as a model for identifying substances increasing colorectal cancer (CRC). To test this possibility, we treated such rats with the bile acid (BA) cholic acid (CA) (0.3% w/w in the diet), known to promote CRC, and assessed tumorigenesis. Precancerous colonic lesions (mucin-depleted foci) and intestinal tumors were dramatically increased in CA-treated rats compared to controls (p<0.01). Colon mucosa proliferation was higher and apoptosis lower than those in controls. Expression of nuclear receptor 1h4 (Nr1h4) gene [encoding for BA receptor farnesoid X receptor (FXR)], organic solute transporter beta (Ostb) and fatty acid-binding protein 6 (Fabp6), FXR-dependent BA transporters, were dramatically down-regulated in CA-treated rats. CA-increased tumorigenesis in female PIRC rats, with mechanisms involving increased proliferation, reduced apoptosis and marked down-regulation of genes controlling BA homeostasis. Since BAs have been implicated in CRC, we suggest that female PIRC rats can be used to identify CRC-promoting agents.

Conserved serum protein biomarkers associated with growing early colorectal adenomas

A major challenge for the reduction of colon cancer is to detect patients carrying high-risk premalignant adenomas with minimally invasive testing. As one step, we have addressed the feasibility of detecting protein signals in the serum of patients carrying an adenoma as small as 6-9 mm in maximum linear dimension. Serum protein biomarkers, discovered in two animal models of early colonic adenomagenesis, were studied in patients using quantitative mass-spectrometric assays. One cohort included patients bearing adenomas known to be growing on the basis of longitudinal computed tomographic colonography. The other cohort, screened by optical colonoscopy, included both patients free of adenomas and patients bearing adenomas whose risk status was judged by histopathology. The markers F5, ITIH4, LRG1, and VTN were each elevated both in this patient study and in the studies of the Pirc rat model. The quantitative study in the Pirc rat model had demonstrated that the elevated level of each of these markers is correlated with the number of colonic adenomas. However, the levels of these markers in patients were not significantly correlated with the total adenoma volume. Postpolypectomy blood samples demonstrated that the elevated levels of these four conserved markers persisted after polypectomy. Two additional serum markers rapidly renormalized after polypectomy: growth-associated CRP levels were enhanced only with high-risk adenomas, while PI16 levels, not associated with growth, were reduced regardless of risk status. We discuss biological hypotheses to account for these observations, and ways for these signals to contribute to the prevention of colon cancer.