Intestinal Tumor Multiplicity Research Articles

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 <500ppm and LFA9 <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<0.0001), while LFA-9 treated male rats had 31% less tumors (22.13±1.19; p<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<0.0002) while LFA-9 treatment led to 37% inhibition (11.89±0.86; p<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.

Abstract 14: TRAIL inducing small molecule ONC201 prevents intestinal tumors in FAP mouse model

Abstract Colorectal cancer (CRC) is a major health problem globally. Preventing CRC in the general population, and especially in high-risk cohorts with colonic polyps, or its recurrence, is a high priority. ONC201, an orally active small molecule with robust antitumor activity, is currently in clinical trials for advanced cancers. The aim of this study was to determine the CRC preventive potential of ONC201 using the Apcmin/+ mouse model for CRC in familial adenomatous polyposis. First, the optimal and non-toxic doses of ONC201 were determined. C57BL/6J mice (n=6/group) were randomized, and five different doses of ONC201 (0-100mg/kg BW) were administered orally twice a week by gavage. After six weeks of treatment, all mice were euthanized, and tissues were analyzed for optimal dose and toxicity. Based on the body weight gain, gross organ weights at termination, blood profiling, plasma liver enzyme profile, and histopathological analysis, it was concluded that there were no toxicities associated with ONC201 within the tested dose range. For efficacy evaluation, Apcmin/+ male and female mice (n≥20) were grouped and ONC201 (0, 25 and 50 mg/kg BW) was administered by gavage, twice weekly for 14 weeks. At termination, intestinal tumors were evaluated for incidence and multiplicity to determine the efficacy of ONC201. Results indicate a strong suppressive effect of ONC201 against colonic and small intestinal (SI) tumor multiplicity in mice of both genders. Colonic tumor incidence was significantly reduced by >50% in ONC201-treated (50 mg/kg BW) male (40% in treated vs. 92% in control; p<0.0002) and female mice (30% in treated vs. 81% in control; p<0.001) when compared with placebo. Colonic tumor multiplicity was reduced by 68% in ONC201-treated (50 mg/kg BW) male mice (0.44 ± 0.11 in treated vs. 1.4 ± 0.14 in control; p<0.0001) and by 75% in female mice (0.30 ± 0.10 in treated vs. 1.19 ± 0.19 in control; p<0.0003). SI polyps were suppressed by up to 68% in both male mice (11.40 ± 1.19 in treated vs. 36.08 ± 2.62 in control; p<0.0001) and female mice (9.65 ± 1.15 in treated vs. 29.24 ± 2.51 in control; p<0.0001). Snap-frozen colon tumor samples were analyzed for various biomarkers. Western blot protein analysis of the tumors suggested a dose-dependent increase in TRAIL expression upon treatment with ONC201. Similar increases were observed for DR5 and cleaved caspase 8 protein expression. Cleaved caspase 7, caspase 3, FADD and p21 were found to be increased in the treated tumor samples when compared with control. Immunohistochemical analysis indicated a significant decrease in PCNA index in the ONC201-treated tumors compared with controls. Serum analysis indicated a decrease in pro-inflammatory biomarkers, such as IL1β, IL6, TNFα, G-CSF, and GM-CSF, in the ONC201-treated samples compared to controls. Overall, chronic oral administration of ONC201 demonstrated strong chemopreventive efficacy against intestinal tumorigenesis in the Apcmin/+ mouse model. (Supported by NCI HHSN 261201500038I) Citation Format: Chinthalapally V. Rao, Venkateshwar Madka, Yuting Zhang, Gopal Pathuri, Janani Panneerselvam, Nicole Stratton, Niklas K. Finnberg, Howard P. Safran, Jennifer Fox, Shizuko Sei, Elizabeth R. Glaze, Robert Shoemaker, Wafik S. El-Deiry. TRAIL inducing small molecule ONC201 prevents intestinal tumors in FAP mouse model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 14.

Usefulness of Capsule Endoscopy and Double-balloon Enteroscopy for the Diagnosis of Multiple Carcinoid Tumors in the Small Intestine: Case Reports and a Literature Review

The incidence of carcinoid tumor in the small intestine is increasing; however, its preoperative diagnosis is difficult. We recently experienced three cases of multiple carcinoid tumors in the small intestine successfully detected using capsule endoscopy (CE), followed by a pathological diagnosis using double-balloon enteroscopy (DBE). To diagnose multiple carcinoid in the small intestine appropriately, we reviewed the information of five cases reported to date along with our three recent cases. The literature review demonstrated that CE and DBE are useful for detecting and diagnosing small intestinal carcinoids and tumor multiplicity, which aids in determining the appropriate resection range.

Abstract 4076: Gut microbiota and metabolite-driven phenotype modulation in a mouse model of colorectal cancer

Abstract Colorectal cancer (CRC) is a multifactorial disease that develops as a result of multiple genetic mutations, often including loss of the tumor suppressor Adenomatous Polyposis Coli (APC) gene. More recently, additional factors such as the host gut microbiota (GM) have also demonstrated an important role in tumor initiation and progression, yet our understanding of most commensal bacteria remains limited. We observed significant differences in intestinal tumor multiplicity between two ApcMin sub-strain mouse colonies; C57BL/6J-ApcMin (B6-Min/J) mice from The Jackson Laboratory had relatively lower adenoma burden while C57BL/6JD-ApcMin (B6-Min/D) mice from the University of Wisconsin develop twice the number of adenomas. To determine the underlying causes of this phenotypic variability, we used complex microbiota targeted rederivation (CMTR) to rederive embryos of the two Apc mutant sub-strains onto surrogate dams harboring distinct GM from two different vendors (GMJAX and GMHSD), generating four ApcMin groups: B6-Min/JGMJAX, B6-Min/JGMHSD, B6-Min/DGMJAX, and B6-Min/DGMHSD. We observed significant increases in intestinal tumor multiplicity in both B6-Min groups harboring the GMHSD compared to their GMJAX counterparts, suggesting that the GM accounts for a significant portion of variability in tumor phenotype between the ApcMin sub-strains. We characterized the GMJAX and GMHSD microbial populations using 16S rRNA gene sequencing to identify potentially causative and protective bacterial taxa. Relative abundances of several taxa including Bilophila sp., Desulfovibrio sp., and Akkermansia muciniphila, were significantly elevated in GMHSD suggesting pro-tumorigenic roles, while the family Peptococcaceae was more abundant in GMJAX, indicating a potential protective role. To establish the functional output of these distinct GM profiles contributing to tumor development, we used liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) to evaluate differences in the intestinal metabolome of the four experimental groups. Notably, we found that the microbiome differences due to colonization with either GMJAX or GMHSD primarily accounted for distinct metabolomic profiles, whereas host genetic differences between the sub-strains had a minimal role in shaping the metabolomes. We detected 31 putative metabolites that were significantly modulated by the GM in both the B6-Min/J and B6-Min/D sub-strains, and were therefore predictive of the relative tumor burden. Moving forward, these metabolites have implications for both biomarker discovery and potential therapeutic and preventative targets. Overall, these results provide insight regarding environmental factors influencing tumorigenesis, and further demonstrate how host-environment interactions can modulate a given disease phenotype. Citation Format: Jacob E. Moskowitz, Zhentian Lei, Susheel Busi, Marcia Hart, Craig Franklin, Lloyd Sumner, James Amos-Landgraf. Gut microbiota and metabolite-driven phenotype modulation in a mouse model of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4076.

Abstract B42: Safer approaches to colorectal cancer chemoprevention

Abstract Colorectal cancer (CRC) protective effects of NSAIDs and COX-2 inhibitors are well established; however, they are associated with gastrointestinal (GI) bleeding and enhanced cardiovascular risk. Mechanistic studies suggest that upregulation 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. One unexplored area in colorectal cancer chemoprevention towards safer drug development is limited or no studies on targeting mPGES-1/5LOX. To design and develop selective inhibitors of dual mPGES-1/5-LOX, we used in silico small-molecular docking simulations approaches, and identified LFA-9 as a novel dual mPGES-1/5-LOX inhibitor among >35 analogs. Azoxymethane (AOM)-induced rat colonic tumors were utilized to assess pharmacodynamic inhibitory effects of LFA-9 on mPGES-1 and 5-LOX by Radio-HPLC. At low μM LFA-9 inhibited the mPGES-1 and 5-LOX activities by ~57% and ~68%, respectively. We tested five different doses (100-1,600 ppm) of LFA-9 by modified AIN-76A diet on male C57 mice and observed that <1,600 ppm dietary LFA-9 was found to be safer and free from the liver and GI toxicities. In AOM/DSS-induced colonic inflammation in F344 rat, 200 ppm and above LFA-9 completely abolishes the colonic inflammation and suppresses mPGES-1 and 5-LOX in dose-response manner. Potential CRC preventive efficacy of LFA-9 was assessed AOM-rat carcinogenesis (Aberrant Crypt Foci, (ACF) as surrogate marker in male F344 rats and intestinal tumor inhibition in APCMin/+ mice. In rats, colonic ACF were induced by AOM/DSS treatment, dietary LFA-9 (200, 400, and 600 ppm) showed dose-response (P<0.0001) inhibitory effects on ACFs formation. In APCMin/+ mice study, diet containing 0, 350, and 700 ppm LFA-9 for 14 weeks administration significantly (p<0.0001) reduced total intestinal tumor multiplicity and size dose-dependently in both male and female mice. At high dose both male and female mice showed > 80% suppression of colonic polyps with >2 mm size. Importantly, mice fed with 350-ppm LFA9 showed colon tumor inhibition of 60% (male) and 90% (female). It is noteworthy that both male and female mice fed with 700-ppm LFA-9 showed 91% inhibition of colon tumors. Overall, the above results show that LFA-9 is a novel dual mPGES-1/5-LOX inhibitor, a safer agent and potential for prevention of CRC in high-risk patients. (Grant support: NCI N01-CN 25001-26 and 1R01CA213987-01.) Citation Format: Chinthalapally V. Rao, Altaf Mohammed, Naveena B. Janakiram. Safer approaches to colorectal cancer chemoprevention [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B42.

Lack of chemopreventive efficacy of metformin in rodent models of urinary bladder, head and neck, and colon/intestine cancer.

Metformin is a biguanide employed in treating type II diabetes. Its potential efficacy for treating cancer has been demonstrated epidemiologically (lower cancer incidence in metformin users compared with users of sulfonylureas or insulin) and mechanistically, primarily in cell culture. Metformin decreases the levels of insulin-like growth factor 1 and secondarily inhibits the mammalian target of rapamycin pathway to exhibit anticancer effects. The current study examined its cancer preventive efficacy in multiple standard in situ arising cancer models. Metformin was administered orally by gavage or in the diet, at human equivalent doses, in numerous cancer models. In the hydroxybutyl(butyl)nitrosamine-induced model of invasive urinary bladder cancer, metformin (50 or 150 mg/kg body weight/day, intragastric) was ineffective despite high urinary concentrations of metformin. Metformin (250 or 500 ppm in diet) failed to decrease the incidence or invasiveness of squamous cell cancer of the tongue in a 4-nitroquinoline-1-(4NQO)-induced model. Finally, in the Min mouse model of gastrointestinal cancer, metformin (400 or 1,200 ppm in diet) was ineffective. Notably, a slight increase in intestinal tumor multiplicity was observed at the higher dose. Therefore, metformin lacked efficacy in multiple standard cancer models in non-diabetic rodents. This lack of efficacy may discourage any large phase clinical cancer trials in non-diabetic individuals in the absence of clear phase-II studies.

ADAM17 is a Tumor Promoter and Therapeutic Target in Western Diet-associated Colon Cancer.

Epidermal growth factor receptors (EGFR) are required for tumor promotion by Western diet. The metalloprotease, ADAM17 activates EGFR by releasing pro-EGFR ligands. ADAM17 is regulated by G-protein-coupled receptors, including CXCR4. Here we investigated CXCR4-ADAM17 crosstalk and examined the role of ADAM17 in tumorigenesis. We used CXCR4 inhibitor, AMD3100 and ADAM17 inhibitor, BMS566394 to assess CXCR4-ADAM17 crosstalk in colon cancer cells. We compared the expression of CXCR4 ligand, CXCL2, and ADAM17 in mice fed Western diet versus standard diet. Separately, mice were treated with marimastat, a broad-spectrum ADAM17 inhibitor, or AMD3100 to assess EGFR activation by ADAM17 and CXCR4. Using Apc-mutant Min mice, we investigated the effects of ADAM17/10 inhibitor INCB3619 on tumorigenesis. To assess the effects of colonocyte ADAM17, mice with ADAM17 conditional deletion were treated with azoxymethane (AOM). ADAM17 expression was also compared in colonocytes from primary human colon cancers and adjacent mucosa. CXCL12 treatment activated colon cancer cell EGFR signals, and CXCR4 or ADAM17 blockade reduced this activation. In vivo, Western diet increased CXCL12 in stromal cells and TGFα in colonocytes. Marimastat or AMD3100 caused >50% reduction in EGFR signals (P < 0.05). In Min mice, INCB3619 reduced EGFR signals in adenomas and inhibited intestinal tumor multiplicity (P < 0.05). In the AOM model, colonocyte ADAM17 deletion reduced EGFR signals and colonic tumor development (P < 0.05). Finally, ADAM17 was upregulated >2.5-fold in human malignant colonocytes. ADAM17 is a Western diet-inducible enzyme activated by CXCL12-CXCR4 signaling, suggesting the pathway: Western diet→CXCL12→CXCR4→ADAM17→TGFα→EGFR. ADAM17 might serve as a druggable target in chemoprevention strategies. Clin Cancer Res; 23(2); 549-61. ©2016 AACR.

Design and validation of an orally administrated active L. fermentum-L. acidophilus probiotic formulation using colorectal cancer Apc Min/+ mouse model.

Probiotics have been shown to have beneficial properties in attenuating the risk of colorectal cancer (CRC) development. However, functional evidence to support such effects for some probiotic bacteria are relatively unknown. Here, we document a significant antioxidant, anti-proliferative and pro-apoptotic activities of Lactobacillus acidophilus ATCC 314 and Lactobacillus fermentum NCIMB 5221 on CRC cells, particularly when used in combination (La-Lf). Furthermore, a superior synergistic activity on the inhibition of tumor growth and modulation of cell proliferation and epithelial markers in the Apc Min/+ CRC mouse model was explored, based on the expression levels of Ki-67, E-cadherin, β-catenin, and cleaved caspase-3 (CC3) proteins. The anti-cancer activity of La-Lf co-culture was significantly enhanced in vitro with significant reduced proliferation (38.8±6.9%, P=0.009) and increased apoptosis (413 RUL, P<0.001) towards cancer cells, as well as significant protection of normal colon cell growth from toxic treatment (18.6±9.8%, P=0.001). La-Lf formulation (1010cfu/animal/day) altered aspects of intestinal tumorigenesis by significantly reducing intestinal tumor multiplicity (1.7-fold, P=0.016) and downregulating cellular proliferation markers, including β-catenin (P=0.041) and Ki-67 (P=0.008). In conclusion, La-Lf showed greater protection against intestinal tumorigenesis supporting a potential use as a biotherapeutic for the prevention of CRC.

Abstract 5248: ED-71, an analogue of Vitamin D3, blocks the promotion but not the initiation of colorectal tumors in the Apc+/Min-FCCC mouse model

Abstract Results from several studies demonstrate an inverse relationship between circulating levels of Vitamin D and risk of colorectal cancer. Eldecalcitol (1α,25-dihydroxy-2β-(3-hydroxypropyloxy) Vitamin D3; ED-71) is a novel analog of calcitriol, the most active form of Vitamin D3. ED-71 is more potent in stimulating bone remodeling and has been approved in Japan for the treatment of osteoporosis. The goal of this study was to assess the ability of ED-71 to inhibit spontaneous colorectal adenomas in a unique strain of multiple intestinal neoplasia (Apc+/Min-FCCC) mice. Male mice (6 wks of age) were randomized to treatment groups based on colon tumor status (endoscopic confirmation as tumor-free or -bearing) and body weight (bw) and administered: vehicle (MCT), calcitriol (0.25 μg/kg bw), or ED-71 (0.05 or 0.1 μg/kg bw) by gavage. Mice were treated every other day and bws were recorded weekly. After 14 wks of treatment, the small intestines and colons were excised and examined for gross tumors. Colorectal tumors &amp;gt;3 mm in diameter were frozen for gene expression analyses. The remaining tissue was fixed in formalin and processed for histological review. The multiplicity of gross small intestinal tumors in animals treated with ED-71 (both doses) was comparable to that of vehicle-treated controls. In contrast, the multiplicity of small intestinal tumors was elevated 34.6% over that of controls in animals treated with calcitriol (Mean ± SEM: 28.4 ± 2.6 vs. 21.1 ± 2.8, respectively; P = 0.034). The ability of ED-71 to inhibit colon tumorigenesis was evaluated independently in mice with vs. without tumors at treatment initiation. In tumor-bearing mice, neither calcitriol nor ED-71 (both doses) had any significant effect on the multiplicity of colorectal tumors as compared to control mice. However in tumor-free mice, ED-71 (0.1 μg/kg bw) reduced the mean incidence of adenomas (&amp;gt; 4 crypts) by 47% (ED-71 - 45.5%, controls 92.3%; P = 0.02) and the multiplicity by 47.6% (Mean ± SEM: ED-71 - 1.1 ± 0.39, controls - 2.1 ± 0.72; P &amp;gt; 0.05). In contrast, the multiplicity of microadenomas (≤ 4 crypts) was increased in mice treated with ED-71 (0.1 μg/kg bw) as compared to controls (0.9 ± 0.22 vs. 0.5 ± 0.25 P = 0.06, respectively). These findings suggest that ED-71 is effective in preventing the transition of microadenomas to mature adenomas. The lack of an effect of ED-71 on colon tumor development in mice bearing tumors at baseline could be due in part to loss of the Vitamin D receptor in colon adenomas, as confirmed by real-time PCR. Insight into the mechanism by which ED-71 inhibits adenoma development is being gained from analyses of Vitamin D signaling and microRNA expression in treated colonic epithelial cells. These promising data provide support for future studies to determine the potential utility of ED-71 in preventing colorectal cancer in high-risk patients found to be tumor-free during surveillance endoscopy. (Supported by NCI HHSN261201200015I) Citation Format: Wen-Chi L. Chang, Harry S. Cooper, Esther Kaunga, Lisa Vanderveer, Jing Peng, Suen S. Chen, Margie L. Clapper. ED-71, an analogue of Vitamin D3, blocks the promotion but not the initiation of colorectal tumors in the Apc+/Min-FCCC mouse model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5248.

Abstract 2604: Chemoprevention of colorectal cancer by LFA-9, a novel dual mPGES-1/5-LOX inhibitor: safer approaches to chemoprevention

Abstract Colorectal cancer (CRC) protective effects of NSAIDs and COX-2 inhibitors are well established; however, they are associated with gastrointestinal (GI) bleeding and cardiovascular risk. Mechanistic studies suggest that sparing COX-1/2, prostaglandin (PG)I2 synthase and selectively targeting microsomal PG synthase-1 (mPGES-1) and 5-lipoxygenase (5-LOX) would inhibit PGE2 and Leukotrienes (LTs), respectively. To design and develop selective inhibitors of mPGES-1/5-LOX, we used insilico small molecular docking simulation approaches, and identified LFA-9 as a novel selective dual mPGES-1/5-LOX inhibitor among &amp;gt;20 analogs. Azoxymethane (AOM)-induced rat colonic tumors were utilized to assess inhibitory effects of LFA-9 on mPGES-1 and 5-LOX ex-vivo. At 7.5 μM, LFA-9 inhibited the mPGES-1 and 5-LOX activities by ∼57% and ∼68%, respectively. Maximum tolerable dose (100 -1,600ppm) of LFA-9 in AIN-76A diet on male C57Bl/6 mice were tested and observed that &amp;lt;1,600 ppm dietary LFA-9 to be safe and free from liver, GI and hematological toxicities. In AOM/DSS-induced colonic inflammation in rat, LFA-9 at ≥200ppm abolished the inflammation and suppressed mPGES-1 and 5-LOX activities in a dose-response manner. Potential CRC preventive efficacy of LFA-9 was assessed in AOM-rat carcinogenesis with colonic Aberrant Crypt Foci, (ACF) as surrogate marker in male F344 rats and intestinal tumor inhibition in APCMin/+ mice. In rats, colonic ACF were induced by AOM/DSS treatment and two week after the AOM/DSS treatment, LFA-9 (200, 400, and 600ppm) was fed by diet and ACF were evaluated after six-weeks. LFA-9 showed dose-response (P&amp;lt;0.0001) inhibitory effect on ACFs formation. At 600ppm, LFA-9 significantly inhibited colonic total ACF and multi-crypts by &amp;gt;60% (P&amp;lt;0.0001). In APCMin/+ mice study, six-week-old male and female mice (10/group) were fed diet containing 0, 350, and 700 ppm LFA-9 for 14 weeks. Male and female APCMin/+ mice fed control diet developed 58±3.8 (Mean±SEM) and 59±5.9 polyps, respectively. LFA-9 administration at 350 and 700ppm in APCMin/+ mice significantly (p&amp;lt;0.0001) reduced total intestinal tumor multiplicity and size dose-dependently (31.6± 5.9 and 24.3±3.7, male mice; and 26.2±5.6 and 15.5±1.8, female mice, respectively). At the high dose both male and female mice showed a &amp;gt; 80% suppression of polyps with &amp;gt;2mm size. Mice fed 350ppm LFA9 showed colon tumor inhibition of 60% (male) and 90% (female). It is noteworthy that both male and female mice fed 700ppm LFA-9 showed 91% inhibition of colon tumors. LFA-9 showed significant suppression of markers of proliferation and inflammation. Overall, above results suggest LFA-9 as a novel dual mPGES-1/5-LOX inhibitor, a safer agent than other NSAIDs and has a potential for prevention of CRC in high-risk patients. (Grant Support: NCI N01-CN 25001-26). Citation Format: Naveena B. Janakiram, Altaf Mohammed, Gopal Pathuri, Venkateshwar Madka, Rebekah Ritchie, Taylor Bryant, Yuting Zhang, Qian Li, Stan Lightfoot, Hariprasad Gali, Steele E. Vernon, Chen S. Suen, Chinthalapally V. Rao. Chemoprevention of colorectal cancer by LFA-9, a novel dual mPGES-1/5-LOX inhibitor: safer approaches to chemoprevention. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2604.

Pegylation Improves the Pharmacokinetics and Bioavailability of Small-Molecule Drugs Hydrolyzable by Esterases: A Study of Phospho-Ibuprofen

Esterase hydrolysis of drugs can accelerate their elimination, thereby limiting their efficacy. Polyethylene glycol (PEG) covalently attached to drugs (pegylation) is known to improve the efficiency of many drugs. Using as a test agent the novel phospho-ibuprofen (PI), we examined whether pegylation of PI could abrogate its hydrolytic degradation by esterases; PI, known to inhibit colon cancer growth, has a carboxylic ester hydrolyzable by carboxylesterases (CES). We covalently attached mPEG-2000 to PI (PI-PEG) and studied its stability by exposing it to cells overexpressing CES and by administering it to mice. We also evaluated PI-PEG's anticancer efficacy in human colon cancer xenografts and in Apc(min/+) mice. PI-PEG was stable in the presence of cells overexpressing CES1 or CES2, whereas PI was extensively hydrolyzed (90.2 ± 0.7%, 14.3 ± 1.1%, mean ± S.E.M.). In mice, PI was nearly completely hydrolyzed. Intravenous administration of PI-PEG resulted in significant levels in blood and in colon cancer xenografts (xenograft values in parentheses): area under the curve for 0-24 hours = 2351 (2621) (nmol/g) × h; Cmax = 1965 (886) nmol/g; Tmax = 0.08 (2) hour. The blood levels of ibuprofen, its main hydrolytic product, were minimal. Compared with controls, PI-PEG inhibited the growth of the xenografts by 74.8% (P < 0.01) and reduced intestinal tumor multiplicity in Apc(min/+) mice by 73.1% (P < 0.01), prolonging their survival (100% versus 55.1% of controls; P = 0.013). Pegylation protects PI from esterase hydrolysis and improves its pharmacokinetics. In preclinical models of colon cancer, PI-PEG is a safe and efficacious agent that merits further evaluation.

Adoptive transfer of regulatory T cells promotes intestinal tumorigenesis and is associated with decreased NK cells and IL‐22 binding protein

High number of regulatory T cells (Tregs), both circulating and at the tumor site, often indicates a poor prognosis in CRC patient's possibly impairing natural killer (NK) cell function. To determine the role of Tregs in CRC development and their effects on NK cells, we created novel transgenic Rag-Apc mice that lack T cells and develop spontaneous intestinal tumors, and we adoptively transferred Tregs or transiently depleted NK cells during initial stages of tumorigenesis. In 6-weeks old Rag-Apc mice containing microscopic intestinal tumors adoptive transfer of Tregs or transient NK cell depletion dramatically associated with an increase in intestinal tumor multiplicity and tumor size, with significantly decreased survival rates. Importantly, Treg transfer increased small intestinal polyp formation up to 65% (P < 0.0005) and increased colon tumors multiplicities by 84% (P < 0.0001) with a significant decrease in NK cells as compared to control mice. Similarly, in NK depleted mice, colon tumor multiplicities increased up to 40% and small intestinal polyp formation up to 60% (P < 0.0001). Treg transfer or NK cell transient depletion markedly increased interleukin (IL)-22 systemically and the inflammatory signaling molecules P2X7R, and STAT3 in the tumors; and impaired production of the tumor suppressor interferon (IFN)-γ systemically. Notably, IL-22 binding protein (IL-22 BP) was associated with NKs and a significant decrease was seen at the tumor site in mice adoptively transferred with Tregs or depleted of NK cells. Our results suggest that adoptive transfer of Tregs aggressively promote intestinal tumorigenesis by decreasing NK cell number and activity by modulating IL-22 BP.

Sa1958 Serrated Polyposis Syndrome: Pushing Through Prevalence and Clinicopathological Features

G A A b st ra ct s diet alone group (p<0.05). INCB3619 significantly inhibited pEGFR and pErbB2 signals in intestinal adenomas. Colon tumor incidence was 0.33 in the INCB3619-treated group, compared to 0.83 in Western diet alone group (p=0.10). Summary INCB3619 significantly reduced intestinal tumor multiplicity and tumor size in Apc Min mice. INCB3619 also concomitantly suppressed intestinal mucosal ADAM17 enzyme activity as well as EGFR signals in intestinal adenomas. Conclusions Our results show that targeting ADAM17 inhibits intestinal tumor promotion byWestern diet.We postulate thatWestern diet promotes tumorigenesis by activating ADAM17 and up-regulating EGFR signals. Our studies suggest that ADAM17 inhibitors might provide a novel strategy to reduce colon cancer risk in individuals consuming Western diets.

Raloxifene and Antiestrogenic Gonadorelin Inhibits Intestinal Tumorigenesis by Modulating Immune Cells and Decreasing Stem-like Cells

Studies suggest that estrogen plays a contributing role in colorectal cancer. This project examined the preventive effects of raloxifene, a selective estrogen receptor modulator (SERM), and gonadorelin, an antiestrogenic drug, in female Apc(Min/+) mouse intestinal tumorigenesis. Six-week-old Apc(Min/+)mice were fed diet containing 1 ppm raloxifene or control diet. Gonadorelin (150 ng/mouse) was injected subcutaneously into one treatment group. Intestinal tumors were evaluated for tumor multiplicity and size. Mice treated with raloxifene and gonadorelin showed colon tumor inhibition of 80% and 75%, respectively. Both drugs significantly inhibited small intestinal tumor multiplicity and size (75%-65%, P < 0.0001). Raloxifene and gonadorelin showed significant tumor inhibition with 98% and 94% inhibition of polyps >2 mm in size. In mice fed with raloxifene or injected with gonadorelin, tumors showed significantly reduced proliferating cell nuclear antigen expression (58%-65%, P < 0.0001). Raloxifene treatment decreased β-catenin, cyclin D1, laminin 1β, Ccl6, and stem-like cells (Lgr 5, EpCAM, CD44/CD24), as well as suppressed inflammatory genes (COX-2, mPGES-1, 5-LOX,). Gonadorelin showed significant decrease in COX-2, mPGES-1, iNOS, and stem-like cells or increased NK cells and chemokines required for NK cells. Both drugs were effective in suppressing tumor growth albeit with different mechanisms. These observations show that either suppression of estrogen levels or modulation of estrogen receptor dramatically suppresses small intestinal and colonic tumor formation in female Apc(Min/+) mice. These results support the concept of chemoprevention by these agents in reducing endogenous levels of estrogen or modulating ER signaling.

High-Energy Particle-Induced Tumorigenesis Throughout the Gastrointestinal Tract

Epidemiological data reveals the gastrointestinal (GI) tract as one of the main sites for low-LET radiation-induced cancers. Importantly, the use of particle therapy is increasing, but cancer risk by high-LET particles is still poorly understood. This gap in our knowledge also remains a major limiting factor in planning long-term space missions. Therefore, assessing risks and identifying predisposing factors for carcinogenesis induced by particle radiation is crucial for both astronauts and cancer survivors. We have previously shown that exposure to relatively high doses of high-energy (56)Fe ions induced higher intestinal tumor frequency and grade in the small intestine of Apc(Min/+) mice than γ rays. However, due to the high number of spontaneous lesions (∼30) that develop in Apc(Min/+) animals, this Apc mutant model is not suitable to investigate effects of cumulative doses <1 Gy, which are relevant for risk assessment in astronauts and particle radiotherapy patients. However, Apc(1638N/+) mice develop a relatively small number of spontaneous lesions (∼3 per animal) in both small intestine and colon, and thus we propose a better model for studies on radiation-induced carcinogenesis. Here, we investigated model particle radiation increases tumor frequency and grade in the entire gastrointestinal tract (stomach and more distal intestine) after high- and low-radiation doses whether in the Apc(1638N/+). We have previously reported that an increase in small intestinal tumor multiplicity after exposure to γ rays was dependent on gender in Apc(1638N/+) mice, and here we investigated responses to particle radiation in the same model. Phenotypical and histopathological observations were accompanied by late changes in number and position of mitotic cells in intestinal crypts from animals exposed to different radiation types.

Abstract A100: Helicobacter hepaticus contributes to mammary gland carcinogenesis through bacterial translocation and subsequent expansion of cancer-promoting myeloid-derived suppressor cells

Abstract Introduction: Gastrointestinal (GI) tract dysbiosis and resulting inflammation are implicated in breast carcinogenesis. CD11b+GR1+ myeloid- derived suppressor cells (MDSCs) are important drivers of breast cancer. Here we report that in APCMin/+ mice treated with enteric Helicobacter hepaticus (H. hepaticus) MDSCs cells are dramatically expanded and, along with the pathogen H. hepaticus, are directly recruited to mammary gland lymph node, where they suppress anti-carcinogenic cytotoxic T-cells and promote procarcinogenic inflammation, respectively. Methods: In our H. hepaticus and specific pathogen free colony we inoculated female APCMin/+ mice with H. hepaticus and compared neoplastic and inflammatory outcomes to the non-infected controls. Results: Successful infection was confirmed by H. hepaticus specific qPCR and fluorescence in situ hybridization (FISH). H. hepaticus was identified not only within the small intestine but also within the mammary gland lymph node and mammary tumor tissue of the treated group. In APCMin/+ mice treated with chow diet, H. hepaticus-induced procarcinogenic CD11b+GR1+ cells within the lamina propria of the small intestine, the surrounding mesenteric lymph nodes, the spleen, peripheral blood and the mammary gland lymph nodes. In H. hepaticus infected mice, this was accompanied by increased splenic expression of IL-1β, IL-6 and GM-CSF and the increased expression of IL-1β, IL-6 and GM-CSF, IL-23a and IL-17a in the small intestine. Interestingly, adding high fat diet to H. hepaticus infected mice appeared to further promote the trafficking of MDSCs from the spleen into the peripheral blood and regional lymph nodes with increased serum inflammatory cytokines production cytokines, including IL-6. This appears to have been mediated, at least in part, by adipocyte signaling to myeloid precursors. Conclusions: We show the presence of H. hepaticus in mammary gland and intestine, which correlated with mammary gland tumor incidence and increased intestinal tumor multiplicity. The co-existence of enteric H. hepaticus infection appears to promote mammary tumorigenesis through upregulation of IL-1β, IL-6 and GM-CSF and the resulting trafficking of tumor promoting CD11b+GR1+ MDSCs. We are currently examining the route and broader relevance of H. hepaticus extra-intestinal translocation and whether this has direct effects on tumorigenesis, consistent with findings in other Helicobacter spp. Citation Format: Xiaowei Chen, Daniel L. Worthley, Zhongming Ge, Yagnesh Tailor, Christian Kaufman, Lenzie Cheaney, Lesley Kline, Gladys Y. Valeriano, Christoph B. Westphalen, Susan E. Erdman, James G. Fox, Timothy C. Wang. Helicobacter hepaticus contributes to mammary gland carcinogenesis through bacterial translocation and subsequent expansion of cancer-promoting myeloid-derived suppressor cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A100.

Multitargeted Low-Dose GLAD Combination Chemoprevention: A Novel and Promising Approach to Combat Colon Carcinogenesis

Preclinical studies have shown that gefitinib, licofelone, atorvastatin, and α-difluoromethylornithine (GLAD) are promising colon cancer chemopreventive agents. Because low-dose combination regimens can offer potential additive or synergistic effects without toxicity, GLAD combination was tested for toxicity and chemopreventive efficacy for suppression of intestinal tumorigenesis in adenomatous polyposis coli (APC)Min/+ mice. Six-week-old wild-type and APCMin/+ mice were fed modified American Institute of Nutrition 76A diets with or without GLAD (25 + 50 + 50 + 500 ppm) for 14 weeks. Dietary GLAD caused no signs of toxicity based on organ pathology and liver enzyme profiles. GLAD feeding strongly inhibited (80–83%, P < .0001) total intestinal tumor multiplicity and size in APCMin/+ mice (means ± SEM tumors for control vs GLAD were 67.1 ± 5.4 vs 11.3 ± 1.1 in males and 72.3 ± 8.9 vs 14.5 ± 2.8 in females). Mice fed GLAD had >95% fewer polyps with sizes of >2 mm compared with control mice and showed 75% and 85% inhibition of colonic tumors in males and females, respectively. Molecular analyses of polyps suggested that GLAD exerts efficacy by inhibiting cell proliferation, inducing apoptosis, decreasing β-catenin and caveolin-1 levels, increasing caspase-3 cleavage and p21, and modulating expression profile of inflammatory cytokines. These observations demonstrate that GLAD, a novel cocktail of chemopreventive agents at very low doses, suppresses intestinal tumorigenesis in APCMin/+ mice with no toxicity. This novel strategy to prevent colorectal cancer is an important step in developing agents with high efficacy without unwanted side effects.

Inhibition of ENNG-Induced Pyloric Stomach and Small Intestinal Carcinogenesis in Mice by High Temperature- and Pressure-Treated Garlic

High temperature- and pressure-treated garlic (HTPG) has been shown to have enhanced antioxidative activity and polyphenol contents. Previously, we reported that HTPG inhibited 1,2-dimethylhydrazine-induced mucin depleted foci (premalignant lesions) and O6-methylguanine DNA adduct formation in the rat colorectum. In the present study, we investigated the modifying effects of HTPG on N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG)- induced pyloric stomach and small intestinal carcinogenesis in mice. Male C57BL/6 mice were given ENNG (100 mg/l) in drinking water for the first 4 weeks, then a basal diet or diet containing 2% or 5% HTPG for 30 weeks. The incidence and multiplicity of pyloric stomach and small intestinal (duodenal and jejunal) tumors in the 2% HTPG group (but not in the 5% HTPG group) were significantly lower than those in the control group. Cell proliferation of normal-appearing duodenal mucosa was assessed by MIB-5 immunohistochemistry and shown to be significantly lower with 2% HTPG (but again not 5% HTPG) than in controls. These results in dicate that HTPG, at 2% in the diet, inhibited ENNG-induced pyloric stomach and small intestinal (especially duodenal) tumorigenesis in mice, associated with suppression of cell proliferation.

Sex-dependent Differences in Intestinal Tumorigenesis Induced in Apc1638N/+ Mice by Exposure to γ Rays

The purpose of the present study was to assess the effect of 1 and 5 Gy radiation doses and to investigate the interplay of gender and radiation with regard to intestinal tumorigenesis in an adenomatous polyposis coli (APC) mutant mouse model. Apc1638N/+ female and male mice were exposed whole body to either 1 Gy or 5 Gy of γ rays and euthanized when most of the treated mice became moribund. Small and large intestines were processed to determine tumor burden, distribution, and grade. Expression of proliferation marker Ki-67 and estrogen receptor (ER)-α were also assessed by immunohistochemistry. We observed that, with both 1 Gy and 5 Gy of γ rays, females displayed reduced susceptibility to radiation-induced intestinal tumorigenesis compared with males. As for radiation effect on small intestinal tumor progression, although no substantial differences were found in the relative frequency and degree of dysplasia of adenomas in irradiated animals compared with controls, invasive carcinomas were found in 1-Gy- and 5-Gy-irradiated animals. Radiation exposure was also shown to induce an increase in protein levels of proliferation marker Ki-67 and sex-hormone receptor ER-α in both non tumor mucosa and intestinal tumors from irradiated male mice. We observed important sex-dependent differences in susceptibility to radiation-induced intestinal tumorigenesis in Apc1638N/+ mutants. Furthermore, our data provide evidence that exposure to radiation doses as low as 1 Gy can induce a significant increase in intestinal tumor multiplicity as well as enhance tumor progression in vivo.

Chemopreventive efficacy of Targretin in rodent models of urinary bladder, colon/intestine, head and neck and mammary cancers

The chemopreventive efficacy of Targretin was evaluated in various rodent cancer models. In the rat model of 4-hydroxybutyl(butyl)nitrosamine (OH-BBN)-induced urinary bladder cancer, it was found that Targretin administered in the diet (beginning one week after the last OH-BBN treatment) for 5.5 months increased the number and size of urinary bladder cancers. In the azoxymethane (AOM)-induced model of colon carcinogenesis (in which rats develop minimally invasive colonic cancers), Targretin was ineffective as a chemopreventive agent, decreasing neither tumor incidence nor multiplicity. Treatment of Min mice with Targretin for 45 days similarly failed to decrease the multiplicity of small intestinal tumors. Similarly, no preventive efficacy was noted for Targretin when the incidence of tumors in the head and neck model (squamous cell tongue tumors) induced by 4-nitroquinoline 1-oxide (4-NQO) were examined. In contrast, use of even a suboptimal dose of Targretin (40ppm) in a sensitive breast cancer model [methylnitrosourea (MNU)-induced ER+ mammary cancers] reduced cancer multiplicity by 60%. Finally, based on the hypothesis that Targretin may decrease the expression of COX‑2, the effects of Targretin and COX inhibitors were compared in these models. There was minimal overlap of efficacy. That is, models which were relatively susceptible to NSAIDs or COX-2 inhibitors tended not to be sensitive to Targretin and vice versa.