Reduced Tumor Multiplicity Research Articles

Abstract 4048: Comparative effects of SOS1 inhibitor with IL-6 blockade and their combination on Kras mutant lung cancer

Abstract Despite recent advances in treatment, lung adenocarcinoma (LUAD), particularly harboring Kras mutations, is still the leading cause of cancer mortality. We and others have previously shown that Kras mutant LUAD development and progression is associated with intrinsic inflammatory characteristics driven by epithelial NF-κB activation, increased levels of IL-6 (which is transcriptionally regulated by NF-κB), and activation of the IL-6-responsive transcription factor STAT3. We further demonstrated that IL-6 blockade suppresses lung tumorigenesis by reducing tumor-associated immunosuppressive myeloid populations and induction of a T cell-mediated anti-tumor response. The recent discovery of a novel SOS1-targeting compound (BI-3406) that selectively inhibits the KRAS-SOS1 interaction and leads to anti-proliferative activity has opened up the possibility to more efficiently inhibit Kras mutant lung cancer. Accordingly, in this study we used a mouse model of KRAS mutant LUAD (CC-LR) to compare the therapeutic effects of BI-3406, IL-6 blockade, and MEK inhibitor (trametinib). We also tested whether inhibiting IL-6 driven pro-tumor immune response would provide a synergistic/additive response and increase the tumor inhibitory effect of either BI-3406 or Trametinib. Eight cohorts of 14-week-old female mice were treated with either anti-IL-6 antibody, trametinib, or BI-3406, or their combinations for 4 weeks, and their tumor burden was compared at the end of study. We observed a significant reduction in tumor multiplicity and tumor area in response to all monotherapies, with the highest effect in the trametinib treated cohort. Neither combination therapy led to decreased tumor burden over either of the monotherapy regimens in our model. However, we observed an increase in T helper 1 and CD8 T expressing IFNγ cell populations in the cohort treated with Trametinib and anti-IL-6 Ab combination. In addition, using qPCR analysis of RNA extracted from the whole lung we found a higher expression of IFNγ along with a reduced expression of IL-10 in the cohort treated with BI-3406 and anti-IL-6 Ab combination. This suggests that these combinations may have the potential to improve the anti-tumor immune responses. Hence, we conclude that further adjustment of the treatment regimen (doses, interval, duration) may allow to obtain improved anti-tumor efficacy with these combinations. Overall, this study provides a new research perspective for the improvement of currently available treatment modalities for patients with Kras mutant lung cancer that could be further explored preclinically. Citation Format: Katherine E. Larsen, Maria J. Arredondo Sancristobal, Melody Zarghooni, Avantika Krishna, Maria T. Grimaldo, Nastaran Karimi, Arnav Gaitonde, Michael J. Clowers, Seyed Javad Moghaddam. Comparative effects of SOS1 inhibitor with IL-6 blockade and their combination on Kras mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4048.

The Chemoprevention Effects of Two Herbal Mixtures on Chemically Induced Lung Tumorigenesis in Mice.

Ruan Hua Tang (RHT) and Ruan Hua Fang (RHF) are two Chinese herbal mixtures that have been used in clinical cancer treatment for decades. This study validated our hypothesis that RHT and RHF can inhibit lung tumor development in the mouse model of Benzo(a)pyrene-induced lung tumorigenesis. An RHT oral solution was diluted to 9% and 18% in water. RHF was mixed into the diet at 15% and 30% of total food in the final doses. Two weeks after injecting BP into mice, we administered RHT and RHF for eighteen weeks. We found that 9% and 18% RHT reduced tumor multiplicity by 36.05% and 38.81% (both p < 0.05) and the tumor load by 27.13% and 55.94% (p < 0.05); 15% and 30% RHF inhibited tumor multiplicity by 12.75% and 39.84% (p < 0.01) and the tumor load by 18.38% and 61.68% (p < 0.05). Ki67 expressions in the 9% and 18% RHT groups were 19.55% and 11.51%, significantly lower than in the control (33.64%). The Ki67 levels in the 15% and 30% RHF groups were 15.56% and 14.04%, significantly lower than in the control (27.86%). Caspase 3 expressions in the 9% and 18% RHT groups were 5.24% and 7.32%, significantly higher than in the control (2.39%). Caspase 3 levels in the 15% and 30% RHF groups were 6.53% and 4.74%, significantly higher than in the control (2.07%). The bio-absorption was confirmed via a pharmacokinetic test. This study showed that RHT and RHF are safe and can inhibit lung tumor development, with anti-proliferative and pro-apoptotic effects.

Abstract 652: Tumor cell-specific IL-1/IL-1R signaling promotes KRAS mutant lung tumorigenesis

Abstract Despite improved diagnosis and treatment strategies such as immunotherapy, lung cancer is still the leading cause of cancer-related deaths worldwide in both men and women. Lung adenocarcinoma (LUAD) with driver mutations in the KRAS oncogene is the most prevalent molecular subtype of lung cancer. KRAS mutant LUAD exhibits aggressive biology in part due to enhanced pro-tumor inflammation mediated by activation of the nuclear factor-κB (NF-κB) and, consequently, elevated expression of various cytokines. While the pro-inflammatory cytokine IL-1β is a product of the NF-κB pathway it also acts as a potent activator, further amplifying the production of protumor cytokines via a positive feedback loop. We have shown that IL-1β blockade enhances anti-tumor immune responses while inhibiting immunosuppression in a mouse model of KRAS mutant LUAD driven by lung epithelial cell-specific expression of KRASG12D (CCSPCre/LSL-KRASG12D, CC-LR mouse). Cell lineage-specific mechanisms that underlie these anti-tumor effects following inhibition of IL-1β are still poorly understood. To fill this void, we here explored the effects of targeting the IL-1R receptor in KRAS mutant lung epithelial cells in the CC-LR mouse model. We studied tumor development and host immune response in 14-week-old CC-LR mice with conditional knockout of IL-1R in KRAS mutant lung epithelial cells (LR/IL-1RΔ/Δ) in comparison to age- and sex-matched control CC-LR littermates. LR/IL-1RΔ/Δ mice displayed markedly reduced tumor multiplicity (~50%) when compared to control CC-LR mice concomitant with decreased cell proliferation and angiogenesis evidenced by attenuated immunohistochemical expression of Ki-67 and ERG. Flow cytometry analysis showed an elevated inflammatory response, most evidently seen through the significant increase in infiltrating monocytes. Interestingly, a shift in type-1 conventional dendritic cells, commonly involved in antigen-cross presentation in tumorigenesis, to type-2 conventional dendritic cells was seen in LR/IL-1RΔ/Δ mice, suggesting a different method of cross-presentation resulting from the conditional knockout of IL-1R that could potentially lead to a protective T-cell response. These results were further confirmed via gene expression analysis of respective markers. Our findings provide further support for the role of the IL-1 cytokine family in the development and progression of KRAS mutant LUAD as well as warrant further studies targeted towards understanding the mechanistic effects IL-1β has on the tumor microenvironment. Citation Format: Avantika Krishna, Michael J. Clowers, Bo Yuan, Milind Mutala, Maria Jose Arredondo Sancristobal, Jocelynn Colunga, Ryan De Maleki, Linda Phan, Humam Kadara, Seyed Javad Moghaddam. Tumor cell-specific IL-1/IL-1R signaling promotes KRAS mutant lung tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 652.

Dietary Supplementation with Black Raspberries Altered the Gut Microbiome Composition in a Mouse Model of Colitis-Associated Colorectal Cancer, although with Differing Effects for a Healthy versus a Western Basal Diet.

Black raspberries (BRB) are rich in anthocyanins with purported anti-inflammatory properties. However, it is not known whether dietary supplementation would ameliorate Western-diet enhanced gut inflammation and colon tumorigenesis. We employed a mouse model of colitis-associated colorectal cancer (CAC) to determine the effects of dietary supplementation with 5 to 10% (w/w) whole, freeze-dried BRB in male C57BL/6J mice fed either a standard healthy diet (AIN93G) or the total Western diet (TWD). In a pilot study, BRB suppressed colitis and colon tumorigenesis while also shifting the composition of the fecal microbiome in favor of taxa with purported health benefits, including Bifidobacterium pseudolongum. In a follow-up experiment using a 2 × 2 factorial design with AIN and TWD basal diets with and without 10% (w/w) BRB, supplementation with BRB reduced tumor multiplicity and increased colon length, irrespective of the basal diet, but it did not apparently affect colitis symptoms, colon inflammation or mucosal injury based on histopathological findings. However, BRB intake increased alpha diversity, altered beta diversity and changed the relative abundance of Erysipelotrichaceae, Bifidobacteriaceae, Streptococcaceae, Rikenellaceae, Ruminococcaceae and Akkermansiaceae, among others, of the fecal microbiome. Notably, changes in microbiome profiles were inconsistent with respect to the basal diet consumed. Overall, these studies provide equivocal evidence for in vivo anti-inflammatory effects of BRB on colitis and colon tumorigenesis; yet, BRB supplementation led to dynamic changes in the fecal microbiome composition over the course of disease development.

Assessment of antitumor activity of BP-C1, a platinum-based anticancer agent with a lignin-derived polymeric ligand, in autochthonous induced and spontaneous carcinogenesis rodent models

BackgroundA standard approach to study the anticancer activity of novel drugs is their testing in animals with inoculated tumors, which has some limitations. An alternative is the use of spontaneous or carcinogen-induced tumor models as they have better translation potential. The carcinogen-induced and transgenic tumor models were used to assess the antitumor activity of BP-C1, a platinum-containing drug with lignin-derived polymeric ligand. MethodsWe used female Swiss-H-derived mice and Wistar female rats to induce autochthonous tumors via exposure to benzo[a]pyrene and 1,2-dimethylhydrazine, respectively. Additionally, transgenic HER-2/neu FVB/N female mice, prone to the development of spontaneous mammary carcinomas, were used. ResultsAntitumor activity of BP-C1 was observed in soft tissue sarcomas, induced by benzo[a]pyrene. The animals treated with BP-C1 exhibited more stabilizations and therapy responses compared to placebo controls. The efficacy of BP-C1 was somewhat reduced compared to cyclophosphamide; however, their combination resulted in an enhanced antitumor effect. For the 1,2-dimethylhydrazine-induced rat colon cancer model, BP-C1 reduced tumor multiplicity by 21–41 %. For mammary adenocarcinomas in HER-2/neu FVB/N mice, short-termed complete responses were observed in the BP-C1 groups with a frequency of 12–13 %, while complete responses were absent in the placebo group. ConclusionThe results acquired indicated a wide spectrum of antitumor activity of BP-C1.

Combined Supplementation with Vitamin B-6 and Curcumin is Superior to Either Agent Alone in Suppressing Obesity-Promoted Colorectal Tumorigenesis in Mice

BackgroundObesity increases the colorectal cancer risk, in part by elevating colonic proinflammatory cytokines. Curcumin (CUR) and supplemental vitamin B-6 each suppress colonic inflammation. ObjectivesWe examined whether the combination of CUR and vitamin B-6 amplifies each supplement's effects and thereby suppress obesity-promoted tumorigenesis. MethodsMale Friend Virus B (FVB) mice (4-week-old; n = 110) received 6 weekly injections of azoxymethane beginning 1 week after arrival. Thereafter, they were randomized to receive a low-fat diet (10% energy from fat), a high-fat diet (HFD; 60% energy from fat), a HFD containing 0.2% CUR, a HFD containing supplemental vitamin B-6 (24 mg pyridoxine HCl/kg), or a HFD containing both CUR and supplemental vitamin B-6 (C + B) for 15 weeks. Colonic inflammation, assessed by fecal calprotectin, and tumor metrics were the primary endpoints. The anti-inflammatory efficacy of the combination was also determined in human colonic organoids. ResultsHFD-induced obesity produced a 2.6-fold increase in plasma IL-6 (P < 0.02), a 1.9-fold increase in fecal calprotectin (P < 0.05), and a 2.2-fold increase in tumor multiplicity (P < 0.05). Compared to the HFD group, the C + B combination, but not the individual agents, decreased fecal calprotectin (66%; P < 0.01) and reduced tumor multiplicity and the total tumor burden by 60%–80% (P < 0.03) in an additive fashion. The combination of C + B also significantly downregulated colonic phosphatidylinositol-4,5-bisphosphate 3-kinase, Wnt, and NF-κB signaling by 31%–47% (P < 0.05), effects largely absent with the single agents. Observations that may explain how the 2 agents work additively include a 2.8-fold increased colonic concentration of 3-hydroxyanthranillic acid (P < 0.05) and a 1.3-fold higher colonic concentration of the active coenzymatic form of vitamin B-6 (P < 0.05). In human colonic organoids, micromolar concentrations of CUR, vitamin B-6, and their combination suppressed secreted proinflammatory cytokines by 41%–93% (P < 0.03), demonstrating relevance to humans. ConclusionsIn this mouse model, C + B is superior to either agent alone in preventing obesity-promoted colorectal carcinogenesis. Augmented suppression of procancerous signaling pathways may be the means by which this augmentation occurs.

Alkaloidal extract from Carica papaya seeds ameliorates CCl4-induced hepatocellular carcinoma in rats

BackgroundHepatocellular carcinoma (HCC) is the third cause of cancer-related mortality globally. However, available treatments are expensive and are associated with adverse effects or poor treatment outcomes in advanced disease. Meanwhile, plants like Carica papaya have demonstrated various biological activities that further studies may lead to the identification of newer and safer treatment options for HCC. AimTo evaluate the anticancer activity of an alkaloidal extract derived from Carica papaya seeds using rodent models of HCC. Experimental procedureCarica Papaya fruits were collected and authenticated. The seeds were isolated and air-dried. Alkaloidal extract was prepared from a 70% ethanol soxhlet crude extract and referred to as Carica papaya alkaloidal extract (CPAE). HCC was induced in 68 out of 84 healthy male Sprague Dawley rats by intraperitoneal injection of carbon tetrachloride (CCl4) for 16 weeks. These rats were put into five groups of 10; Carica papaya alkaloidal extract [(CPAE) (50, 100, and 200 mg/kg), Lenvatinib (4 mg/kg)], 1% dimethyl sulphoxide (DMSO), and 2 untreated groups (control and model). A prophylaxis study was performed with 10 rats by co-administration of CPAE (200 mg/kg) and CCl4 six hours apart for 16 weeks. Rats were sacrificed after a twelve-week treatment program under anesthesia for histological, hematological, and biochemical analyses. Results and conclusionCPAE (100 and 200 mg/kg) significantly restored weight loss (48.44 and 51.75% respectively), reduced tumor multiplicity, and dose-dependently reversed liver histomorphological changes induced by CCl4 compared to the model group. The CPAE (100 and 200 mg/kg) further reduced bleeding time, improved prothrombin time and restored platelet count (p < 0.01) compared to the model. The CPAE (200 mg/kg) again significantly (p < 0.0001) reduced serum alpha-fetoprotein levels compared to the model group and prevented the establishment of HCC in rats when concerrently administered with CCl4 in 16 weeks prophylactic study.

Loss of Peroxiredoxin IV Renders Mice Resistant to Chemically Induced Colon Carcinogenesis

Oxidative stress is an important contributing factor in many types of human disease including cancer. One of the major antioxidants involved in the maintenance of cellular redox balance is the Peroxiredoxin (Prx) family of proteins. They carry out such function through scavenging and reducing hydrogen peroxide, alkyl hydroperoxides and peroxynitrite with high sensitivity. Peroxiredoxin IV (Prx4) is one of the six members of the Prx family and is highly conserved in prokaryotes and eukaryotes. In human genome, Prx4 gene is located in the X chromosome (Xp22.11). Prx4 protein is mainly localized in the Endoplasmic Reticulum (ER) where it plays an important role in ROS scavenging as well as oxidative protein folding. It is also present in the cytoplasm and can be secreted into extracellular space under physiological conditions. Sulfiredoxin (Srx) is another enzyme that protects cells against oxidative stress by reducing hyperoxidized 2-cysteine-containing Prxs. Prx4 has been found to be overexpressed in a number of human cancers including colorectal cancer. Previously, we have discovered that reduced expression of Srx disrupts chemically induced colorectal cancer development in mice. However, the role of Prx4 in colon carcinogenesis has not been studied before. In this study, Prx4 null mice and Srx-Prx4 null mice were generated in pure FVB/N background. Mouse colon carcinogenesis was induced by azoxymethane (AOM) and dextran sulfate sodium (DSS) protocol. Briefly, wildtype, Prx4 null mice and Srx-Prx4 null mice at 8-week of age were injected intraperitoneally with 10mg/kg of AOM in week 1. Mice were administered with two rounds of 2% DSS in drinking water in weeks 2 and 10. Twenty weeks after AOM injection, all mice were euthanized and examined for tumors. Our results indicate that Prx4 null mice have significantly reduced tumor multiplicity as well as smaller tumor size. This suggests that Prx4 has a critical oncogenic role in cancer development. We are currently conducting mechanistic studies to determine how the loss of Prx4 leads to mice resistant to colon carcinogenesis in the AOM/DSS model.

Abstract PD11-08: Low dose endoxifen represses mouse mammary tumorigenesis: A preclinical study of monotherapy and combination with ulipristal acetate

Abstract Background: Endoxifen (ENX), a major active metabolite of the selective estrogen receptor (ER) modulator tamoxifen (TAM), has been shown to inhibit the growth of ER+ cancer. However, it has not been evaluated for prevention of mammary cancer. We report here the cancer prevention efficacy of ENX as monotherapy and in combination with ulipristal acetate (UPA), a selective progesterone receptor (PR) modulator. We compare these to TAM using the SV40 C3 T-antigen (C3-TAg) mouse, a well-documented mammary cancer prevention model. Methods: C3-TAg female virgin mice aged 7-8 weeks were randomized to no-treatment control or to drug treatment groups (TAM, ENX, UPA and ENX+UPA). Drug pellets were subcutaneously implanted on backside of mice. The doses of drug pellet were 12.5mg/30d for TAM, 9mg/90d for ENX, 10mg/30d for UPA, and combination of ENX (9mg/90d) + UPA (10mg/30d). The dose of ENX was equivalent to the exposure resulting from 20 mg/day, the lowest dose used in a recent Phase I human trial. Pellets were replaced every 30 days or 90 days. Mammary tumor formation was monitored twice weekly by palpation. Tumor latency, multiplicity, and tumor volume were recorded; animals are euthanized at 23 weeks of age, or earlier if tumors reach &amp;gt; 1 cm2. Tumors and mammary glands were formalin fixed and paraffin embedded for histology evaluation by a rodent pathologist. The primary endpoint was a reduction in tumor incidence in drug treated versus control groups. Secondary endpoints included prolongation of latency, reduction in tumor multiplicity, and tumor burden. Statistical significance between groups was calculated with Wilcoxon log-rank test for % tumor-free survival and Mann Whitney test for tumor multiplicity and burden.Results: All mice in the control group developed tumors by 18 weeks of age. Tumor-free survival % of ENX, UPA, and ENX+UPA treated groups were significantly higher than control animals, with the greatest increase in the ENX group (p=0.02). TAM had no effect on tumor-free survival (p=0.32). Median tumor latencies were similar in three treated groups: ENX (112 days), UPA (114 days), and ENX+UPA (111 days) groups, and were significantly delayed compared to the control or TAM group (100 days in both) (p&amp;lt;0.05). Median tumor multiplicity (invasive adenocarcinoma) per animal was significantly lower in ENX group compared to control group (3 vs. 7, p=0.02). UPA and TAM did not significantly reduce tumor multiplicity (4 for TAM and 6 for UPA). We observed non-significant 32% and 23% reduction in median tumor weight of ENX+UPA group and ENX group, respectively compared to control group. Histology evaluation for ENX+UPA group and pathologic size of invasive carcinoma is ongoing, along with the expression of Ki67, ERα and PR of mammary tumors and mammary gland by immunohistochemistry.Conclusions: TAM showed no significant effect on delaying tumorigenesis, consistent with a previous study. However, low dose of ENX (equivalent to 24 mg/day in human) effectively repressed tumor development and growth than TAM or UPA treatment. Our data suggest that ENX is promising for prevention of ER+ mammary tumorigenesis and warrant a dose optimization study for improving efficacy. Citation Format: Oukseub Lee, Minhua Wang, Maarten C. Bosland, Omid Hosseini, Irene Helenowski, Seema A. Khan. Low dose endoxifen represses mouse mammary tumorigenesis: A preclinical study of monotherapy and combination with ulipristal acetate [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD11-08.

Alkaloidal Extract From &lt;i&gt;Carica papaya&lt;/i&gt; Seeds Ameliorates CCl &lt;sub&gt;4&lt;/sub&gt;-Induced Hepatocellular Carcinoma in Rats

Background: Hepatocellular carcinoma (HCC) is the third cause of cancer-related mortality globally, however, available treatments are expensive and associated with adverse effects or poor treatment outcomes in advanced disease. Meanwhile, plants like Carica papaya have demonstrated hepatoprotective effects, and further studies may lead to the identification of newer and safer treatment options for HCC. Aim: To evaluate the anticancer activity of an alkaloidal extract derived from Carica papaya seeds using rodent models of hepatocellular carcinoma. Experimental Procedure: Carica papaya fruits were collected and authenticated. The seeds were isolated and air-dried. Alkaloidal extract was prepared from a 70% ethanol soxhlet crude extract and referred to as Carica papaya alkaloidal extract (CPAE). HCC was induced in 68 out of 84 healthy male Sprague Dawley rats by intraperitoneal injection of carbon tetrachloride (CCl4) for 16 weeks. These rats were put into five groups of 10; Carica papaya alkaloidal extract [(CPAE) (50, 100, and 200 mg/kg), Lenvatinib (4 mg/kg)], 1% dimethyl sulphoxide (DMSO), and 2 untreated groups (control and model). A prophylaxis study was performed with 10 rats by co-administration of CPAE (200 mg/kg) and CCl4 six hours apart for 16 weeks. Rats were sacrificed under anesthesia for histological, hematological, and biochemical analyses. Results and Conclusion: CPAE (100 and 200 mg/kg) significantly restored weight loss (48.44 and 51.75% respectively), reduced tumor multiplicity, and dose-dependently reversed liver histomorphological changes induced by CCl4 compared to the model group. CPAE (200 mg/kg) also significantly (p < 0.0001) reduced serum alpha-fetoprotein levels compared to the model group. Additionally, CPAE (100 and 200 mg/kg) significantly (p < 0.01) reduced bleeding time, improved prothrombin time, restored normal platelet count, and prevented the establishment of HCC in 16 weeks prophylactic study.

Abstract 3966: The procancer effects of obesity can be reversed by moderate weight loss or an anti-inflammatory drug in a mouse model of colon cancer

Abstract Purpose: Obesity is associated with an increased risk of colon cancer. Our current study examines whether moderate weight loss or treatment with the non-steroidal anti-inflammatory drug sulindac suppresses the tumor-promoting effects of obesity in a mouse model of colon cancer. Methods: Male FVB mice were treated with azoxymethane (10 mg/kg i.p.) for 5 weeks, then randomized to a control (10% kcal from fat) or diet-induced obesity (DIO, 60% kcal from fat) diet. After 15 weeks, there was an interim sacrifice of 5 mice/group. The DIO mice were then further randomized to remain on DIO or switch to control diet to induce weight loss and become formerly obese mice (FOB), and within each diet group (control, DIO, and FOB), half the mice were randomized to start sulindac treatment (140 ppm in the diet). Eight weeks later, all mice were euthanized. Results: The DIO mice, compared to controls, had significantly greater body weight and body fat (P&amp;lt;0.05), while FOB mice had intermediate levels of adiposity. Sulindac did not affect adiposity in any diet group. At the interim timepoint, DIO mice had 10-fold greater tumor multiplicity relative to control mice (P=0.07). At study endpoint, DIO mice had greater tumor multiplicity compared to both control (P&amp;lt;0.05) and FOB (P&amp;lt;0.01) mice, which did not significantly differ from each other. Sulindac reduced tumor multiplicity in both control (P&amp;lt;0.05) and DIO (P&amp;lt;0.0001) mice, but not FOB mice. Serum levels of several inflammation-related proteins, including interleukin 6, chemokine (C-X-C motif) ligand 1, monocyte chemoattractant protein 1, and vascular endothelial growth factor, were elevated in DIO mice versus control (P&amp;lt;0.05) and DIO+sulindac (P&amp;lt;0.01) mice, but not in comparison to FOB mice. Gene expression in paired colon tumor and mesenteric adipose tissue samples was assessed by microarray. Ingenuity pathway analysis (IPA) of the significant (P&amp;lt;0.05) differentially expressed tumor genes indicated that both weight loss and sulindac reduced signaling related to matrix metalloproteinases (MMPs) and inflammation. In the visceral adipose tissue, IPA demonstrated that weight loss reduced signaling related to B cell receptors and glucose/fatty acid metabolism while sulindac decreased MMP, prostaglandin, and PPAR signaling. Additional analyses to explore the potential role of cross-talk between these two tissues are being performed, and key findings will be validated in the tissues at the protein level and via mechanistic cell culture experiments. Conclusions: Both moderate weight loss and sulindac treatment completely reverse the effects of chronic obesity on colon tumorigenesis, the latter independent of any change in adiposity. Our findings suggest that an investigation regarding the effects of NSAID treatment on colon cancer risk and/or progression in obese patients is warranted, particularly for those unable to achieve moderate weight loss. Citation Format: Laura W. Bowers, Arunima Punjala, Alison Q. Jeffries, Stephanie A. Montgomery, Andrew J. Dannenberg, Stephen D. Hursting. The procancer effects of obesity can be reversed by moderate weight loss or an anti-inflammatory drug in a mouse model of colon cancer [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 3966.

Abstract A46: Dietary weight loss and sulindac treatment each reverse obesity-associated inflammation and tumorigenesis in a mouse model of carcinogen-induced colon cancer

Abstract Purpose: We previously showed diet-induced obesity (DIO) increases (and calorie restriction decreases) systemic markers of inflammation and azoxymethane (AOM)-induced colon carcinogenesis in FVB/N mice. Our current study examines whether moderate weight loss via low-fat diet (LFD) and/or treatment with the nonsteroidal anti-inflammatory drug sulindac reverses the inflammatory and tumor-promoting effects of DIO in the AOM-induced mouse model of colon cancer. Methods: Male FVB/N mice were treated with AOM (10 mg/kg i.p.) for 5 weeks, then randomized to a control (10% kcal from fat) or DIO (60% kcal from fat) diet. After 15 weeks on diet, 5 mice/group were killed and their tissues collected for interim timepoint analysis. The remaining DIO mice were further randomized to either remain on DIO or switch to control diet to induce weight loss (formerly obese [FOb] mice), and within each diet group (control, DIO, and FOb), half the mice were randomized to receive sulindac treatment (140 ppm in the diet). Eight weeks later, all mice were killed and their serum, tumors, and other tissues collected for analysis. Results: The DIO mice, compared to controls, had significantly greater body weight and body fat (P&amp;lt;0.05), while FOb mice had intermediate levels of weight and adiposity. Sulindac did not affect adiposity in any diet group. At the interim timepoint, DIO mice had ~10-fold greater tumor multiplicity relative to control mice (p&amp;lt;0.01) and Fob mice (p&amp;lt;0.01). The control and FOb groups did not significantly differ from each other. At study termination sulindac significantly reduced tumor multiplicity in both control (P&amp;lt;0.05) and DIO (P&amp;lt;0.0001) mice, but not FOb mice. In the mice that received sulindac, tumor multiplicity did not significantly differ between diet groups. Tumor multiplicity in the DIO+sulindac, FOb, and FOb+sulindac groups was also significantly lower than the interim timepoint DIO mice (P&amp;lt;0.05), suggesting the interventions induced tumor regression. Serum levels of several inflammation-related proteins, including interleukin 6, chemokine (C-X-C motif) ligand 1, monocyte chemoattractant protein 1, and vascular endothelial growth factor, were elevated in DIO mice versus controls (P&amp;lt;0.05), but did not differ between FOb and control mice. Sulindac treatment significantly reduced serum levels of these proteins in DIO mice (P&amp;lt;0.01). Gene expression in paired colon tumor and mesenteric adipose tissue samples is currently being assessed by microarray analysis to investigate crosstalk between these tissues and inform the design of future studies on the mechanism(s) mediating sulindac’s effects. Conclusions: Both moderate weight loss and sulindac treatment reverse the effects of chronic obesity on AOM-induced colon tumorigenesis. The anticancer effects of sulindac were independent of any change in adiposity. Given the challenges of losing weight, our findings suggest that further investigation of the effects of NSAID treatment on colon cancer risk and/or progression in obese patients is warranted. Citation Format: Stephen D. Hursting, Laura W. Bowers, Arunima Punjala, Andrew J. Dannenberg. Dietary weight loss and sulindac treatment each reverse obesity-associated inflammation and tumorigenesis in a mouse model of carcinogen-induced colon cancer [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A46.

The Combination of Curcumin and Salsalate is Superior to Either Agent Alone in Suppressing Pro‐Cancerous Molecular Pathways and Colorectal Tumorigenesis in Obese Mice

High-fat diets (HFDs) and adiposity increase colorectal cancer risk, in part by elevating pro-inflammatory cytokines that activate pro-cancerous signaling pathways. Curcumin (CUR), a dietary polyphenol and salsalate (SAL), an non-steroidal anti-inflammatory drug (NSAID) lacking the gastrotoxicity of aspirin, each suppress inflammatory signaling, but via different cellular pathways. A/J mice (n=110) are fed a low-fat diet (LFD, 10% kcal), a HFD (60% kcal), a HFD containing 0.4% CUR, a HFD containing 0.3% SAL, or a HFD containing both agents (CUR/SAL). All mice receive six injections of azoxymethane. Compared to LFD-fed mice, HFD-fed mice display elevated colonic cytokines, crypt cell proliferation, and increased tumorigenesis (p < 0.05). CUR/SAL significantly reduces colonic cytokines (p < 0.01), suppresses activation of the PI3K/Akt/mTOR/NF-κB/Wnt pathways (p < 0.01), activates AMPK (p < 0.01), attenuates abnormal proliferation of the colonic mucosa (p < 0.05), and reduces tumor multiplicity and burden (p < 0.05), in comparison to the HFD control. In contrast, CUR or SAL alone does not suppress abnormal crypt cell proliferation or tumor multiplicity, and is largely ineffective in modifying activation of these signaling pathways. These observations demonstrate the superiority of the CUR/SAL over the individual agents and provide a scientific basis for future translational studies in obese subjects and/or those habitually consuming HFDs.

Chemoprevention of Lung Carcinogenesis by Dietary Nicotinamide and Inhaled Budesonide.

Nicotinamide, the amide form of vitamin B3, and budesonide, a synthetic glucocorticoid used in the treatment of asthma, were evaluated to determine their individual and combinational chemopreventive efficacy on benzo(a)pyrene-induced lung tumors in female A/J mice. Nicotinamide fed at a dietary concentration of 0.75% significantly inhibited tumor multiplicity. Nicotinamide by aerosol inhalation at doses up to 15 mg/kg/day did not result in a statistically significant reduction in tumor multiplicity. Finally, dietary nicotinamide was administered with aerosol budesonide and tumor multiplicity reduced by 90% at 1 week and 49% at 8 weeks post last carcinogen dose. We conclude nicotinamide is an effective and safe agent for lung cancer dietary prevention at both early- and late-stage carcinogenesis and that efficacy is increased with aerosol budesonide. Combination chemoprevention with these agents is a well-tolerated and effective strategy which could be clinically advanced to human studies.

Prevention of Wogonin on Colorectal Cancer Tumorigenesis by Regulating p53 Nuclear Translocation.

The tumor suppressor protein p53 plays an important role in the development and progression of colon cancer, and the subcellular organelle localization directly affects its function. Wogonin (5,7-dihydroxy-8-methoxyflavone), a mono-flavonoid extracted from root of Scutellaria baicalensis Georgi, possesses acceptable toxicity and has been used in colorectal cancer (CRC) chemoprevention in pre-clinical trials by oncologist. However, the underlying anti-colon cancer mechanisms of wogonin are not yet fully understood. In the present study, the effect of wogonin on the initiation and development of colitis-associated cancer through p53 nuclear translocation was explored. AOM-DSS CRC animal model and human CRC HCT-116 cell model were used to evaluate the in vivo and in vitro anti-colon cancer action of wogonin. We observed that wogonin showed a dramaticlly preventive effect on colon cancer. Our results showed that wogonin caused apoptotic cell death in human CRC HCT-116 cell through increased endoplasmic reticulum (ER) stress. Meanwhile, excessive ER stress facilitated the cytoplasmic localization of p53 through increasing phosphor-p53 at S315 and S376 sites, induced caspase-dependent apoptosis and inhibited autophagy. Furthermore, we verified the chemoprevention effect and toxicity of wogonin in vivo by utilizing an AOM-DSS colon cancer animal model. We found that wogonin not only reduced tumor multiplicity, preserved colon length to normal (6.79 ± 0.34 to 7.41 ± 0.56, P < 0.05) but also didn’t induce side effects on various organs. In conclusion, these results explain the anti-tumor effect of wogonin in CRC and suggest wogonin as a potential therapeutic candidate for the therapeutic strategy in CRC treatment.

Early increase in blood supply (EIBS) is associated with tumor risk in the Azoxymethane model of colon cancer

BackgroundThe present study aimed to investigate the role of blood supply in early tumorigenesis in colorectal cancer. We leveraged the renin angiotensin system (RAS) to alter colonic blood supply and determine the effect on tumor initiation and progression.MethodsTo test the effect of blood supply on tumorigenesis, 53 male A/J mice were randomly assigned to one of three RAS modulation groups and one of two AOM treatments. The RAS modulation groups were I) water (RAS-unmodulated) as a control group, II) angiotensin-II and III) the angiotensin receptor blocker, Losartan. The mice in each group were then randomly split into either the saline control condition or the AOM-treated condition in which tumors were induced with a standard protocol of serial azoxymethane (AOM) injections. To monitor microvascular changes in the rectal mucosa during the study, we used confocal laser endomicroscopy (CLE) with FITC-Dextran for in-vivo imaging of vessels and polarization-gated spectroscopy (PGS) to quantify rectal hemoglobin concentration ([Hb]) and blood vessel radius (BVR).ResultsAt 12 weeks post-AOM injections and before tumor formation, CLE images revealed many traditional hallmarks of angiogenesis including vessel dilation, loss of co-planarity, irregularity, and vessel sprouting in the pericryptal capillaries of the rectal mucosa in AOM-Water tumor bearing mice. PGS measurements at the same time-point showed increased rectal [Hb] and decreased BVR. At later time points, CLE images showed pronounced angiogenic features including irregular networks throughout the colon. Notably, the AOM-Losartan mice had significantly lower tumor multiplicity and did not exhibit the same angiogenic features observed with CLE, or the increase in [Hb] or decrease in BVR measured with PGS. The AOM-AngII mice did not have any significant trends.ConclusionIn-vivo PGS measurements of rectal colonic blood supply as well as CLE imaging revealed angiogenic disruptions to the capillary network prior to tumor formation. Losartan demonstrated an effective way to mitigate the changes to blood supply during tumorigenesis and reduce tumor multiplicity. These effects can be used in future studies to understand the early vessel changes observed.

Dietary Walnuts Protect Against Obesity-Driven Intestinal Stem Cell Decline and Tumorigenesis.

Obesity can negatively impact intestinal homeostasis, and increase colon cancer risk and related mortality. Thus, given the alarmingly high rates of obesity in the US and globally, it is critical to identify practical strategies that can break the obesity-cancer link. Walnuts have been increasingly recognized to mitigate cancer risk, and contain many bioactive constituents with antioxidant and anti-inflammatory properties that could potentially counteract pathways thought to be initiators of obesity-related cancer. Therefore, the purpose of this study was to determine if walnuts could preserve intestinal homeostasis, and attenuate tumorigenesis and growth in the context of obesity and a high calorie diet. To this end, we studied effects of walnuts on these parameters under different dietary conditions in wildtype mice, two independent Apc models (Apc1638N/+ and ApcΔ14), and in MC38 colon cancer cells in vivo, respectively. Walnuts did not alter the metabolic phenotype or intestinal morphology in normal mice fed either a low-fat diet (LFD), LFD with 6% walnuts (LFD+W), high-fat diet (HFD), or HFD with 7.6% walnuts (HFD+W). However, walnuts did lead to a significant reduction in circulating CCL5 and preserved intestinal stem cell (ISC) function under HFD-fed conditions. Furthermore, walnuts reduced tumor multiplicity in Apc1638N/+ male HFD+W animals, as compared to HFD controls (3.7 ± 0.5 vs. 2.5 ± 0.3; P = 0.015), tended to reduce the number of adenocarcinomas (0.67 ± 0.16 vs. 0.29 ± 0.12; P = 0.07), and preferentially limited tumor growth in ApcΔ14 male mice (P = 0.019) fed a high-calorie western-style diet. In summary, these data demonstrate that walnuts confer significant protection against intestinal tumorigenesis and growth and preserve ISC function in the context of a high-calorie diet and obesity. Thus, these data add to the accumulating evidence connecting walnuts as a potentially effective dietary strategy to break the obesity-colon cancer link.

Mesenchymal MAPKAPK2/HSP27 drives intestinal carcinogenesis

Mesenchymal cells in the microenvironment of cancer exert important functions in tumorigenesis; however, little is known of intrinsic pathways that mediate these effects. MAPK signals, such as from MAPKAPK2 (MK2) are known to modulate tumorigenesis, yet their cell-specific role has not been determined. Here, we studied the cell-specific role of MK2 in intestinal carcinogenesis using complete and conditional ablation of MK2. We show that both genetic and chemical inhibition of MK2 led to decreased epithelial cell proliferation, associated with reduced tumor growth and invasive potential in the Apcmin/+ mouse model. Notably, this function of MK2 was not mediated by its well-described immunomodulatory role in immune cells. Deletion of MK2 in intestinal mesenchymal cells (IMCs) led to both reduced tumor multiplicity and growth. Mechanistically, MK2 in IMCs was required for Hsp27 phosphorylation and the production of downstream tumorigenic effector molecules, dominantly affecting epithelial proliferation, apoptosis, and angiogenesis. Genetic ablation of MK2 in intestinal epithelial or endothelial cells was less effective in comparison with its complete deletion, leading to reduction of tumor size via modulation of epithelial apoptosis and angiogenesis-associated proliferation, respectively. Similar results were obtained in a model of colitis-associated carcinogenesis, indicating a mesenchymal-specific role for MK2 also in this model. Our findings demonstrate the central pathogenic role of mesenchymal-specific MK2/Hsp27 axis in tumorigenesis and highlight the value of mesenchymal MK2 inhibition in the treatment of cancer.

Shared and independent functions of aPKC\u03bb and Par3 in skin tumorigenesis

The polarity proteins Par3 and aPKC are key regulators of processes altered in cancer. Par3/aPKC are thought to dynamically interact with Par6 but increasing evidence suggests that aPKC and Par3 also exert complex-independent functions. Whereas aPKCλ serves as tumor promotor, Par3 can either promote or suppress tumorigenesis. Here we asked whether and how Par3 and aPKCλ genetically interact to control two-stage skin carcinogenesis. Epidermal loss of Par3, aPKCλ, or both, strongly reduced tumor multiplicity and increased latency but inhibited invasion to similar extents, indicating that Par3 and aPKCλ function as a complex to promote tumorigenesis. Molecularly, Par3/aPKCλ cooperate to promote Akt, ERK and NF-κB signaling during tumor initiation to sustain growth, whereas aPKCλ dominates in promoting survival. In the inflammatory tumorigenesis phase Par3/aPKCλ cooperate to drive Stat3 activation and hyperproliferation. Unexpectedly, the reduced inflammatory signaling did not alter carcinogen-induced immune cell numbers but reduced IL-4 Receptor-positive stromal macrophage numbers in all mutant mice, suggesting that epidermal aPKCλ and Par3 promote a tumor-permissive environment. Importantly, aPKCλ also serves a distinct, carcinogen-independent role in controlling skin immune cell homeostasis. Collectively, our data demonstrates that Par3 and aPKCλ cooperate to promote skin tumor initiation and progression, likely through sustaining growth, survival, and inflammatory signaling.

Daily or weekly dosing with EGFR inhibitors, gefitinib and lapatinib, and AKt inhibitor MK2206 in mammary cancer models.

Daily vs. weekly dosing with EGFR inhibitors (gefitinib and lapatinib) and an AKT inhibitor (MK2206) were compared in two rodent breast cancer models. Female Sprague-Dawley rats were administered methylnitrosourea (MNU) at 50 days of age, and gefitinib (daily/weekly dosing at 10/70 mg/kg BW) or lapatinib (daily/weekly dosing at 75/525 mg/kg BW) were administered by gavage beginning 5 days after MNU. For the prevention studies, weekly or daily dosing with gefitinib or lapatinib reduced cancer multiplicity >75%, and all treatments reduced tumor weights by >90%. For the therapeutic studies, MNU-treated rats were followed until small palpable mammary cancers developed. The rats were then treated daily or weekly as above for 6 weeks. Either daily or weekly dosing with lapatinib or gefitinib caused regression in >50% of the tumors. Immunohistochemistry biomarker studies in palpable mammary cancers following a weekly dose of gefitinib showed that 1 day (but not 7 days) after treatment, the levels of phosphorylated EGFR1 were significantly decreased. In an ER-negative (ER−) Neu-overexpressing model employing MMTV-Neu/P53KO mice, daily (100 mg/kg BW/day, 5 days each week), or weekly dosing (500 or 250 mg/kg BW) with gefitinib reduced tumor multiplicity 65, 85 and 75%, respectively. In the MNU prevention model, daily dosing (100 mg/kg BW/day) with the allosteric AKT inhibitor MK2206 was ineffective, while weekly dosing (700 mg/kg BW) reduced the final tumor weight >70%. Combining weekly MK2206 with the aromatase inhibitor vorozole (0.12 mg/kg BW/day) showed that each compound alone reduced tumor multiplicity 40–50%. The combination reduced cancer multiplicity ~70%. These studies demonstrate the efficacy of weekly dosing with various protein kinase inhibitors; raising the possibility of employing these agents in a breast cancer preventive setting.