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Simple Summary Obesity contributes to one-fifth of cancer deaths. Ovarian cancer (OC) progression is frequently asymptomatic, making its early detection difficult and its chance of survival low. OC expresses highly tumorigenic chemokines, CXCL1/8, of which the specific receptor CXCR2 is increased in the adipocytes. So, the CXCL1/8-CXCR2 axis may appear as a molecular link between obesity and OC. Here, we generated adipocyte-specific CXCR2 conditional knockout (cKO) mice to investigate how this CXCR2 cKO affects the peritoneal dissemination of OC under obese conditions. High-fat, diet-induced obese mice had a shorter survival than lean mice. Particularly, obese cKO mice had a reduced tumor burden but increased ascites accumulation, showing a decreased floating tumor burden in ascites, as well as proliferation and macrophage infiltration in tumors compared to obese wild-type mice. Despite the ascites accumulation, adipocyte-specific CXCR2 cKO reduced the obesity-induced tumor burden, likely altering the peritoneal tumor microenvironment of OC. Abstract Obesity contributes to ovarian cancer (OC) progression via tumorigenic chemokines. Adipocytes and OC cells highly express CXCR2, and its ligands CXCL1/8, respectively, indicating that the CXCL1/8-CXCR2 axis is a molecular link between obesity and OC. Here, we investigated how the adipocyte-specific CXCR2 conditional knockout (cKO) affected the peritoneal tumor microenvironment of OC in a high-fat diet (HFD)-induced obese mouse model. We first generated adipocyte-specific CXCR2 cKO in mice: adipose tissues were not different in crown-like structures and adipocyte size between the wild-type (WT) and cKO mice but expressed lower levels of CCL2/6 compared to the obese WT mice. HFD-induced obese mice had a shorter survival time than lean mice. Particularly, obese WT and cKO mice developed higher tumors and ascites burdens, respectively. The ascites from the obese cKO mice showed increased vacuole clumps but decreased the floating tumor burden, tumor-attached macrophages, triglyceride, free fatty acid, CCL2, and TNF levels compared to obese WT mice. A tumor analysis revealed that obese cKO mice attenuated inflammatory areas, PCNA, and F4/80 compared to obese WT mice, indicating a reduced tumor burden, and there were positive relationships between the ascites and tumor parameters. Taken together, the adipocyte-specific CXCR2 cKO was associated with obesity-induced ascites despite a reduced tumor burden, likely altering the peritoneal tumor microenvironment of OC.

Sterol regulatory element-binding protein 1 (SREBP-1), a master transcription factor in lipogenesis and lipid metabolism, is critical for disease progression and associated with poor outcomes in prostate cancer (PCa) patients. However, the mechanism of SREBP-1 regulation in PCa remains elusive. Here, we report that SREBP-1 is transcriptionally regulated by microRNA-21 (miR-21) in vitro in cultured cells and in vivo in mouse models. We observed aberrant upregulation of SREBP-1, fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC) in Pten/Trp53 double-null mouse embryonic fibroblasts (MEFs) and Pten/Trp53 double-null mutant mice. Strikingly, miR-21 loss significantly reduced cell proliferation and suppressed the prostate tumorigenesis of Pten/Trp53 mutant mice. Mechanistically, miR-21 inactivation decreased the levels of SREBP-1, FASN, and ACC in human PCa cells through downregulation of insulin receptor substrate 1 (IRS1)-mediated transcription and induction of cellular senescence. Conversely, miR-21 overexpression increased cell proliferation and migration; as well as the levels of IRS1, SREBP-1, FASN, and ACC in human PCa cells. Our findings reveal that miR-21 promotes PCa progression by activating the IRS1/SREBP-1 axis, and targeting miR-21/SREBP-1 signaling pathway can be a novel strategy for controlling PCa malignancy.

Claudin-4 is part of the Claudin family of transmembrane tight junction (TJ) proteins found in almost all tissues and, together with adherens junctions and desmosomes, forms epithelial and endothelial junctional complexes. Although the distribution of Claudin-4 occurs in many cell types, the level of expression is cell-specific. Claudin proteins regulate cell proliferation and differentiation by binding cell-signaling ligands, and its expression is upregulated in several cancers. As a result, alterations in Claudin expression patterns or distribution are vital in the pathology of cancer. Profiling the genetic expression of Claudin-4 showed that Claudin-4 is also a receptor for the clostridium perfringens enterotoxin (CPE) and that Claudin-4 has a high sequence similarity with CPE’s high-affinity receptor. CPE is cytolytic due to its ability to form pores in cellular membranes, and CPE treatment in breast cancer cells have shown promising results due to the high expression of Claudin-4. The C-terminal fragment of CPE (c-CPE) provides a less toxic alternative for drug delivery into breast cancer cells, particularly metastatic tumors in the brain, especially as Claudin-4 expression in the central nervous system (CNS) is low. Therefore, c-CPE provides a unique avenue for the treatment of breast–brain metastatic tumors.

Acute-phase proteins (APPs) are associated with a variety of disorders such as infection, inflammatory diseases, and cancers. The signature profile of APPs in breast cancer (BC) is poorly understood. Here, we identified serum amyloid A (SAA) for proinflammatory predisposition in BC through the signature profiles of APPs, interleukin (IL) and tumor necrosis factor (TNF) superfamily using publicly available datasets of tumor samples and cell lines. Triple-negative breast cancer (TNBC) subtype highly expressed SAA1/2 compared to HER2, luminal A (LA) and luminal B (LB) subtypes. IL1A, IL1B, IL8/CXCL8, IL32 and IL27RA in IL superfamily and CD70, TNFSF9 and TNFRSF21 in TNF superfamily were highly expressed in TNBC compared to other subtypes. SAA is restrictedly regulated by nuclear factor (NF)-κB and IL-1β, an NF-κB activator highly expressed in TNBC, increased the promoter activity of SAA1 in human TNBC MDA-MB231 cells. Interestingly, two κB-sites contained in SAA1 promoter were involved, and the proximal region (−96/−87) was more critical than the distal site (−288/−279) in regulating IL-1β-induced SAA1. Among the SAA receptors, TLR1 and TLR2 were highly expressed in TNBC. Cu-CPT22, TLR1/2 antagonist, abrogated IL-1β-induced SAA1 promoter activity. In addition, SAA1 induced IL8/CXCL8 promoter activity, which was partially reduced by Cu-CPT22. Notably, SAA1/2, TLR2 and IL8/CXCL8 were associated with a poor overall survival in mesenchymal-like TNBC. Taken together, IL-1-induced SAA via NF-κB-mediated signaling could potentiate an inflammatory burden, leading to cancer progression and high mortality in TNBC patients.

Human Interlekin-17 is produced by memory activated CD4+ T cells and other cells. It was initially considered unique in that its specific receptor is distinct from other cytokine receptors. IL-17 receptor is ubiquitously expressed by different cells including T cells. IL-17 plays a role in regulating growth, immune response and pro-inflammatory responses. It regulates differentiation of a subset of Th0 cells into Th-17 cells, which produce IL-17-induced cytokines. The IL-17R belongs to type 1 cytokine receptors. IL-17 belongs to a superfamily of its own, which includes IL-17A, IL-17B, IL-17C, IL-17E and IL-17F. These members of IL-17 superfamily have some sequence homology but bind to different receptors. Prior to this investigation, limited information existed on the effects of IL-17A in human leukemia cell lines. Our results show that IL-17A promotes growth, anti-apoptotic effects, chemotaxis, cytokine expression and transcriptional factor activation in leukemia cells. IL-17A activates multiple signaling pathways including PI-3 K, Jak–STAT, Raf-ERK1/2 and SRC kinase pathways, which mediate different biological effects of IL-17A in leukemia cells. Our findings implicate IL-17A in leukemia cell growth and survival, supporting potential leukemia therapy via development of anti-IL-17A drugs. This chapter focuses on IL-17A, herein referred to as IL-17.

KDM5B (lysine[K]-specific demethylase 5B) is frequently upregulated in various human cancers including prostate cancer. KDM5B controls H3K4me3/2 levels and regulates gene transcription and cell differentiation, yet the contributions of KDM5B to prostate cancer tumorigenesis remain unknown. In this study, we investigated the functional role of KDM5B in epigenetic dysregulation and prostate cancer progression in cultured cells and in mouse models of prostate epithelium–specific mutant Pten/Kdm5b. Kdm5b deficiency resulted in a significant delay in the onset of prostate cancer in Pten-null mice, whereas Kdm5b loss alone caused no morphologic abnormalities in mouse prostates. At 6 months of age, the prostate weight of Pten/Kdm5b mice was reduced by up to 70% compared with that of Pten mice. Pathologic analysis revealed Pten/Kdm5b mice displayed mild morphologic changes with hyperplasia in prostates, whereas age-matched Pten littermates developed high-grade prostatic intraepithelial neoplasia and prostate cancer. Mechanistically, KDM5B governed PI3K/AKT signaling in prostate cancer in vitro and in vivo. KDM5B directly bound the PIK3CA promoter, and KDM5B knockout resulted in a significant reduction of P110α and PIP3 levels and subsequent decrease in proliferation of human prostate cancer cells. Conversely, KDM5B overexpression resulted in increased PI3K/AKT signaling. Loss of Kdm5b abrogated the hyperactivation of AKT signaling by decreasing P110α/P85 levels in Pten/Kdm5b mice. Taken together, our findings reveal that KDM5B acts as a key regulator of PI3K/AKT signaling; they also support the concept that targeting KDM5B is a novel and effective therapeutic strategy against prostate cancer. Significance: This study demonstrates that levels of histone modification enzyme KDM5B determine hyperactivation of PI3K/AKT signaling in prostate cancer and that targeting KDM5B could be a novel strategy against prostate cancer.

EZH2 is crucial for the progression of prostate cancer (PCa) and castration-resistant prostate cancer (CRPC) through upregulation and activation of progenitor genes, as well as androgen receptor (AR)-target genes. However, the mechanisms by which EZH2 is regulated in PCa and CRPC remain elusive. Here we report that EZH2 is post-transcriptionally regulated by SKP2 in vitro in cultured cells and in vivo in mouse models. We observed aberrant upregulation of Skp2, Ezh2 and histone H3 lysine 27 trimethylation (H3K27me3) in both Pten null mouse embryonic fibroblasts (MEFs) and Pten null mouse prostate tissues. Loss of Skp2 resulted in a striking decrease of Ezh2 levels in Pten/Trp53 double-null MEFs and in prostate tumors of Pten/Trp53 double-null mutant mice. SKP2 knockdown decreased EZH2 levels in human PCa cells through upregulation of TRAF6-mediated and lysine(K) 63-linked ubiquitination of EZH2 for degradation. Ectopic expression of TRAF6 promoted the K63-linked ubiquitination of EZH2 to decrease EZH2 and H3K27me3 levels in PCa cells. In contrast, TRAF6 knockdown resulted in a reduced EZH2 ubiquitination with an increase of EZH2 and H3K27me3 levels in PCa cells. Furthermore, the catalytically dead mutant TRAF6 C70A abolished the TRAF6-mediated polyubiquitination of recombinant human EZH2 in vitro. Most importantly, a concurrent elevation of Skp2 and Ezh2 was found in CRPC tumors of Pten/Trp53 mutant mice, and expression levels of SKP2 and EZH2 were positively correlated in human PCa specimens. Taken together, our findings revealed a novel mechanism on EZH2 ubiquitination and an important signaling network of SKP2-TRAF6-EZH2/H3K27me3, and targeting SKP2-EZH2 pathway may be a promising therapeutic strategy for CRPC treatment.

The American Cancer Society estimates that 1,688,780 new cases of cancer will be diagnosed in 2017 and that 600,920 people will die from their disease. The burden of cancer is disproportionately borne by the poor and underserved. Underrepresented minority physician scientists and clinician researchers are uniquely qualified to address these disparities as they have frequently experienced them in their own families and communities. As some portion of health disparities is rooted in socioeconomic status and lesser education, economically deprived and first generation college students of all races and ethnicities also experience these disparities. Unfortunately, the number of physicians who do research has been declining so the need for programs that educate students in the art and science of research is increasing. The Meharry Medical College Summer Program in Integrative Science and Cancer Research (MMC-SPiISCR) has provided Meharry medical students with the opportunity to participate in short-term cancer research experiences for eleven years. The program is unique in combining weekly half-day workshops at MMC, an historically black college or university, with a research experience supported by faculty mentors at the Vanderbilt University School of Medicine (VUSM),an institution that includes the Vanderbilt Ingram Cancer Center, one of only 69 NCI-Designated Cancer Centers in the United States and District of Columbia. The workshops include topics emphasizing bioethics and responsible conduct of research as well as presentations by Meharry faculty on their cancer research, cancer disparities, cancer and big data and more. The overarching goal of this program is to inspire Meharry medical students to aspire to medical careers in academic medicine and/or careers that will ultimately include cancer research. To date, 73 MMC students have started and completed the program and 94% are from underrepresented minority groups. The male to female ratio reflects the male to female ratio of the class. Over half of the participants presented their work at meetings outside of the program including AACR National Meeting, AACR Science of Cancer Disparities, Society of Black Academic Surgeons, Student National Medical Association, American Medical Association Research Symposium, American Society for Clinical Oncology, KBRIN Bioinformatics Summit and the Meharry-Vanderbilt-TSU Cancer Partnership Annual Retreat. The students are authors on 27 manuscripts and these numbers are still growing as is the interest of program participants in doing a year of research. Surveys of both mentors and program participant mentees overwhelmingly support the strength of this program and participation in the future. This comprehensive program will go far towards fueling the physician-scientist pipeline with researchers whose life experiences mirror those of minority and underserved patients. Citation Format: Dana R. Marshall, Carol Freund-Taylor, Philip Lammers, Leon Dent, Samuel Adunyah, Billy Ballard. Meharry Medical College medical student summer program in integrative science and cancer research [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 5286.

Butyrate is a histone deacetylase inhibitor implicated in many studies as a potential therapy for various forms of cancer. High concentrations of butyrate (>1.5 mM) have been shown to activate apoptosis in several cancer cell lines including prostate, breast, and leukemia. Butyrate is also known to influence multiple signaling pathways that are mediators of cytokine production. The purpose of this study was to evaluate the impact of high concentrations of butyrate on the cancer microenvironment vis-à-vis apoptosis, cellular migration, and capacity to modulate cytokine expression in cancer cells. The results indicate that high concentrations of butyrate induced a 2-fold activation of caspase-3 and reduced cell viability by 60% in U937 leukemia cells. Within 24 hours, butyrate significantly decreased the levels of chemokines CCL2 and CCL5 in HL-60 and U937 cells, and decreased CCL5 in THP-1 leukemia cells. Differential effects were observed in treatments with valproic acid for CCL2 and CCL5 indicating butyrate-specificity. Many of the biological effects examined in this study are linked to activation of the AKT and MAPK signaling pathways; therefore, we investigated whether butyrate alters the levels of phosphorylated forms of these signaling proteins and how it correlated with the expression of chemokines. The results show that butyrate may partially regulate CCL5 production via p38 MAPK. The decrease in p-ERK1/2 and p-AKT levels correlated with the decrease in CCL2 production. These data suggest that while promoting apoptosis, butyrate has the potential to influence the cancer microenvironment by inducing differential expression of cytokines.

Objective. Although hydroxyurea (HU) has been used clinically to treat patients with sickle cell disease (SCD), not all patients benefit from HU treatment due to its toxicity. The objective of this study was to investigate the effectiveness of the use of two new Hb F-inducing nitrosourea analogues, 2-[3-(2-methyl, 2-nitroso) ureido]-2-deoxy-D-glucopyranose (MNGU) and 2-[3-(2-chloroethyl) ureido]-2-deoxy-D-glucopyranose (CGU), in combination with HU in K562 cells or erythroid progenitors. Materials and Methods. After K562 cells were cultured with different concentrations of HU with CGU or MNGU, aliquots of the cells were obtained to determine the total (benzidine-positive) hemoglobin level, number of F cells, and Hb F level. Erythroid progenitor cells of SCD patients and healthy donors were cultured with the optimal drug concentrations, and the number of BFU-E and Hb F level were determined. Results. Our results showed that the combined use of HU with CGU or MNGU increased the number of both benzidine-positive normoblasts and F cells in a synergistic manner. Further, a lower concentration of HU was required to induce a significant level of Hb F synthesis when combined with either of the two compounds in comparison with treatment with HU alone. On day 4, the number of benzidine-positive cells was 4.5- to 6.5-fold and the number of F cells was 5.0- to 8.0-fold higher than the respective numbers in the untreated K562 cells. Similarly, a 3.2- to 14.3-fold induction of Hb F was obtained when human erythroid progenitors from SCD patients were treated with the same drug combinations. Conclusion. Based on these results, the use of CGU or MNGU in combination with HU might offer substantial benefits to patients with SCD and other hemoglobinopathies.

Obesity has become a global epidemic and causes a chronic low-grade inflammation. This poses a large health burden because excess adiposity can lead to chronic diseases such as type 2 diabetes, cardiovascular disease and some cancers. Obese individuals get higher incidence, progression and mortality of cancers compared to lean ones. Particularly, breast cancer (BC) in women is closely associated with obesity. Despite an epidemiological link between obesity and low cancer survival rates, little is known about the molecular mechanisms by which obesity promotes BC progression. Obesity-derived chronic low-grade inflammation involves alteration of a chemokine network that may contribute to the cancer progression and metastasis. Herein, we proposed the novel concept that the chemokine network between obesity and BC builds an inflammatory burden via proinflammatory chemokines to promote cancer progression. We employed mesenchymal BC cell PY8119 and epithelial-like BC PY230 for triple negative BC (TNBC) cell model, and mouse 3T3-L1 preadipocyte and adipocyte conditioned media (CM) to mimic lean and obese conditions in vitro. We examined the profiles of chemokine signature of PY8119 and PY230 treated with preadipocytes and adipocytes CM using PCR array. Both preadipocytes and adipocytes CM-treated PY8119 and PY230 cells absent or low levels in chemokine receptors. On the other hand, the chemokines CCL2, 5, 7 and CXCL10 showed higher expression in both preadipocyte and adipocyte CM-treated PY8119. Interestingly, adipocyte CM-treated PY8119 cells dominantly expressed CXCL1-3 compared to preadipocyte CM-treated cells. In addition, CCL2, 5, 7, 20, and CXCL1, 5, 10 were highly expressed in both preadipocyte and adipocyte CM-treated PY230 cells. Moreover, CXCL2 and 3 were significantly induced in PY230 cells after treatment with adipocyte CM compared to preadipocyte CM. Taken together, the results indicate that dominant chemokines CXCL1-3 expressed in adipocyte CM-treated TNBC cells promote a pro-tumor inflammatory network. Hence, these proinflammatory microenvironment augmented by adipocytes might contribute to TNBC progression. Citation Format: Rosa Mistica Coles Ignacio, Hyeongjwa Choi, Carla Gibbs, Eunsook Lee, Samuel Adunyah, Deok-Soo Son. Involvement of proinflammatory chemokine network between adipocytes and triple negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 950. doi:10.1158/1538-7445.AM2017-950

Enhancer of zeste homolog 2 (EZH2) plays crucial roles on the development of cells and tissues as well as the progression of prostate cancer (PCa) and castration resistant prostate cancer (CRPC). EZH2 can upregulate and activate progenitor genes including androgen receptor (AR) and the target genes. However, the mechanisms by which EZH2 is regulated in PCa remain elusive. Literature reports that tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6) is essential for both normal tissue development and regulation of oncogenic signaling pathways in cancers. In this study, we reported that EZH2 is regulated by PTEN and SKP2 network in PCa. Specifically, we showed that the aberrant upregulation of EZH2 and histone H3 lysine 27 trimethylation (H3K27me3) in both Pten null mouse embryonic fibroblasts (MEFs) in vitro and prostate tumors of Pten null mice in vivo, as compared to the controls. EZH2 levels were negatively correlated with TRAF6 in human PCa cells upon SKP2 dysregulation. Immunofluorescence (IF) staining results showed a co-localization of EZH2 and TRAF6 in nucleus of PC3 cells, and co-immunoprecipitation (co-IP) analysis further confirmed a physical binding of EZH2 and TRAF6 in PC3 cells. Ectopic expression of TRAF6 promoted the K63-linked polyubiquitination of EZH2 to decrease EZH2 and H3K27me3 levels in PCa cells. Conversely, TRAF6 knockdown resulted in a reduction of EZH2 polyubiquitination with an increase of EZH2 and H3K27me3 levels in PCa cells. Furthermore, the catalytically dead mutant TRAF6 C70A abolished the TRAF6-mediated polyubiquitination of human EZH2 in vivo in 293T cells and in vitro in the recombinant human EZH2 protein, as compared with TRAF6 WT. Together, we report for the first time a novel mechanism on EZH2 ubiquitination and an important signaling network of SKP2-TRAF6-EZH2-H3K27me3 in PCa cells. Our findings provide valuable clues on the mechanism and efficiency of targeting EZH2 in PCa and CRPC . Note: This abstract was not presented at the meeting. Citation Format: Wenfu Lu, Shenji Lu, Bo Li, Yingqiu Xie, Michael G. Izban, Billy R. Ballard, Sandeep A. Sathyanarayana, Samuel E. Adunyah, Robert J. Matusik, Zhenbang Chen. EZH2 regulation through lysine 63 linked ubiquitination in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5326. doi:10.1158/1538-7445.AM2017-5326

Breast cancer (BC) is the most frequent tumor in women worldwide. Although its mortality has significantly decreased owing to advanced therapies, triple negative breast cancer (TNBC) is still difficult to treat because of lack of estrogen receptor, progesterone receptor and HER-2. TNBC accounts for 12-24% of total BC and contributes to the aggressiveness and poorer outcomes. The risk of BC increases significantly in obese women. Obesity is also associated with the worse clinical prognosis of BC. But the underlying mechanisms between obesity and BC, particularly TNBC, remain unclear. Therefore, we compare the molecular mechanisms by which human adipocyte conditioned media (CM) affect TNBC cells (BT549) and non-TNBC cells (MCF7A). Adipocyte CM had no effect on the proliferation of both TNBC and non-TNBC cells. However, adipocyte CM enhanced significantly migration in TNBC cells compared to non-TNBC cells. Next, we examined the signaling pathway by which adipocyte CM promote migration of TNBC cell. AKT and ERK were activated in both TNBC cells and non-TNBC cells. However, phospho-STAT3 was significantly increased in TNBC cells. Also, N-cadherin, a marker for epithelial to mesenchymal transition (EMT), was increased at the late time point in TNBC cells. Furthermore, CXCR7 was specifically increased in TNBC cells after treatment with adipocyte CM using a PCR array. CXCR7 is chemoattractive to CXCL12 which is highly expressed in the lung, the bone marrow, and the liver, common sites of BC metastasis. Taken together, the results indicate that adipocyte CM may promote metastasis of TNBC cells through the CXCL12-CXCR7 axis by activating STAT3. Citation Format: Hyeongjwa Choi, Rosa Mistica C. Ignacio, Carla R. Gibbs, Eunsook Lee, Samuel E. Adunyah, Deok-Soo Son. Involvement of CXCL12-CXCR7 axis in adipocyte-induced TNBC metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2933. doi:10.1158/1538-7445.AM2017-2933

Colorectal cancer ranks third in terms of mortalities in the United States. Consumption of Western Diet (rich in red meat and fats), contaminated with environmental toxicants such as benzo(a)pyrene [B(a)P] has also been implicated as one of the causative factors for sporadic colon cancer. Therefore, the objective of this study was to investigate the effect of dietary fat type on B(a)P -induced colon cancer in an adult male rat model, the Polyposis In the Rat Colon (PIRC) kindred type. Groups of PIRC rats (n = 5) were fed with AIN-76A regular diet (RD) or Western diet (WD) and these rats also received 25, 50 and 100 µg B(a)P/kg body wt., daily via oral gavage for a period of 60 days. Rats that were fed with the diets alone, but no B(a)P served as controls. Food consumption and body weights of the rats were periodically monitored. Subsequent to exposure, rats were sacrificed; colons, liver and other tissues were retrieved and preserved in 10% formalin for observation of gross pathological changes. Blood samples were collected and concentrations of cholesterol, triglycerides, and adiponectin were measured. Colon tissues were scored for tumors, and preserved in 10% formalin for observation of pathological changes. Colon and liver samples were analyzed for activation of drug metabolizing enzymes (DMEs) CYP1A1, CYP1B1 and GST. The lack of change in food consumption notwithstanding, body weight loss of WD group compared to RD group and controls (p < 0.05) was noticed. An increased incidence of adenomas and high grade dysplasia were encountered in rats that were fed with WD compared to RD and controls (p < 0.05). The colon tumor counts were more in B(a)P + WD rats compared to their B(a)P + RD counterparts, and also exhibited a B(a)P dose-response relationship, with 100 µg B(a)P/kg registering greater counts. Adenomas with high grade dysplasia were prominent in B(a)P + WD rats compared to B(a)P + RD rats. Immunohistochemical analyses of colon tissue samples for PCNA, cyclin D1, TGF-β, and β-catenin revealed increased levels of cell proliferation and nuclear positivity among all treatment groups. Rats that received B(a)P + WD showed increased levels of cholesterol and triglycerides in comparison to rats that received B(a)P + RD and also controls. Levels of adiponectin did not vary much between B(a)P + WD, and B(a)P + RD groups. Western diet consumption increased DME activation among rats that were given B(a)P + WD with marked increase in rats that were administered 100 µg/kg B(a)P + WD (p < 0.05) compared to other treatment groups. Our results demonstrate that WD accelerates the development of colon tumors induced by B(a)P through proinflammatory action, characterized by gain in tumor number and sizes, and body weight loss. Citation Format: Kelly L. Harris, Stephanie R. Pulliam, Mohammad S. Niaz, Mary K. Washington, Samuel E. Adunyah, Aramandla Ramesh. Western diet enhances benzo(a)pyrene [B(a)P]-induced colon tumorigenesis in the PIRC rat model via proinflammatory mechanisms. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1583. doi:10.1158/1538-7445.AM2014-1583

Sodium butyrate (NaB) is a sodium salt of butyric acid. It is a HDAC inhibitor implicated in many studies as a potential therapy for various forms of cancer. Due to its ability to promote total and gamma globin during erythroid differentiation, low concentrations (1.5 mM) have been shown to activate apoptosis in several cancer cell lines including prostate, endometrial, breast and leukemia. Low concentrations of NaB influence multiple signaling pathways that are known mediators of cytokine production. However, it is not known whether high concentrations alter these pathways having the capacity to modulate cytokine expression in cancer cells and alter the cancer microenvironment, which is the purpose of this study. We exposed U937 leukemia cells to NaB at 5 or 10 mM doses for various time points. The culture media of the cells were harvested and analyzed by Human Multi-Analyte Cytokine ELISArray for levels of chemokines and chemotactic properties via migration assay. As evidence of apoptosis, we monitored cells for caspase-3 activation and cell viability. Our results indicate NaB induces a 2-fold activation of caspase-3 but a decrease in cell viability by 60%. Also, NaB induces a significant decrease in levels of chemokines CCL2 and CCL5 in 24 hours. Moreover, monocyte migration towards culture media from NaB treated cells, decreased in time-dependent manner. To elucidate the signaling mechanism(s) used by NaB (5mM), we investigated whether NaB alters the phospho-protein levels of AKT, ERK1/2, p38 and JNK. We observed an increase in phosphorylation of p38 MAPK, but a decrease in ERK1/2 in response to NaB. Also, there was an initial decrease in p-AKT level within 2 minutes suggesting a decline of Akt activation. However, this was followed by re-activation of AKT by 24 hours post-treatment. Interestingly, NaB did not influence activation of JNK. This data suggests that while promoting apoptosis, NaB has the potential to influence cancer microenvironment by inducing differential expression of cytokines. While the mechanism by which NaB induces differential expression of cytokines remains unknown, potentially, it could occur via the AKT and/or MAPK mediated pathways. These effects may influence the biology of normal and cancer cells thereby altering the status of the patient receiving NaB therapy. *Pulliam is supported by NHLBI T32 Pre-doctoral training grant #2-T32-HL007735-16 to Dr. Adunyah and CTSA award #UL1TR000445 from the National Center for Advancing Translational Sciences. Additionally, Dr. Adunyah is supported by NCI Cancer Partnership U54 grant #5-U54-CA163069-02 and NIMHD MeTRC grant #8-U54-MD007593-04. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. Citation Format: Stephanie R. Pulliam, Roland Cooper, Samuel E. Adunyah. Sodium butyrate suppresses production of chemokines in human U937 cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 142. doi:10.1158/1538-7445.AM2014-142

Sporadic colon cancers have been shown to be triggered by diet and environmental factors. Epidemiological studies have shown that colon cancer rates were significantly reduced in Mediterranean countries where olive oil is the main ingredient of diet. Published works have shown that olive oil inhibits cell proliferation and promotes apoptosis in colorectal cancer cell lines. Studies conducted earlier in our laboratory have shown that dietary exposure to the environmental toxicant benzo(a)pyrene (BaP) leads to colon tumor formation. Benzo(a)pyrene biotransformation studies have shown that once BaP is metabolized by cytochrome P450 drug metabolizing enzymes, it forms metabolites such as BaP 7, 8-diol-epoxide (BPDE), BaP 3,6 & 6,12-diones. These metabolites have also been shown to cause double stranded breaks in DNA and cause oxidative damage. Therefore, we hypothesize that olive oil's interaction with BaP will modulate BaP metabolism and reduce colon tumor formation. In this study we investigated the effect of olive oil on BaP-induced colon carcinogenesis in male ApcMin mice. Mice were assigned to a control (n=7) or treatment group (n =7). Treatment consisted of 50 and 100 μg BaP/kg body weight dissolved in tricaprylin (BaP-only group) or olive oil administered daily via oral gavage for sixty days. Post exposure, mice were sacrificed; colon and liver tissues were retrieved from each group of mice and were preserved in formalin and pathological changes were evaluated. The tissues were further analyzed for the induction of drug metabolizing enzymes such as cytochrome 1A, cytochrome 1B1, and glutathione S-Transferase. Additionally, to determine if BaP caused oxidative DNA damage to tissues, genomic DNA was isolated from the respective tissues and we quantified aldehyde lesions. There was a reduced incidence of adenomas in colons of mice that ingested BaP + olive oil compared to mice that received BaP only (p < 0.05). CYP protein expression was reduced while GST protein expression increased in the colon and liver tissues of BaP + olive oil-treated mice compared to BaP only-treated mice. The BaP organic metabolite concentrations decreased, while aqueous metabolite concentrations increased in the BaP + olive oil group compared to BaP only-treatment group. DNA lesions were increased in the colon and liver tissues of BaP-treated mice compared to BaP + olive oil treated mice. In summary, our studies suggest that olive oil exerts a protective effect against BaP-induced colon cancer. Citation Format: Leah D. Banks, Priscilla Amoah, Mohammad S. Niaz, Mary K. Washington, Samuel E. Adunyah, Aramandla Ramesh. Olive oil alters benzo(a)pyrene biotransformation and reduces oxidative DNA damage in colon of ApcMin mouse. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1589. doi:10.1158/1538-7445.AM2014-1589

Colorectal cancer (CRC) is the third-leading malignancy in the United States. This year alone, about 141,210 new cases of colorectal cancer will be diagnosed and more than 49,380 people will die from the disease. Strategies have focused on early screening practices and the prevention of the development of colonic adenomas that serve as precursors of invasive colon cancers. We have recently showed that chloroquine (CQ) an anti-malarial and anti-rheumatoid agent has the capacity to competitively inhibit intracellular arginase-1 (ARG-1) and that this inhibition increases the nuclear localization of the cell cycle regulator p53 protein in colon cancer cell lines. However, the mechanism of CQ-induced inhibition of ARG-1 that results in the up-regulation of p53 is unclear. The goal of the current study was to examine the chemopreventive efficacy of CQ in the development of colonic neoplasia and to further evaluate the possible mechanism(s) of action. In this regard, six weeks old Apc(Min/+) mice were treated daily (except Saturdays and Sundays) with 0-50 mg CQ/kg body wt. via oral gavage for forty days. Post-treatment, mice were sacrificed and jejunum and colon were retrieved and preserved in 10% formalin for observation of any gross pathological changes. The results of this study showed an increased in prevalence of colonic adenomas in untreated mice compared with treated mice (< 0.05). Interestingly, histochemical analysis showed significant numbers of adenomas with high-grade dysplasia (as evidenced by the presence of increased fibrous tissue core) in untreated control mice in comparison with drug-treated mice ( Citation Format: Efe W. Iyamu, Mohammad S. Niaz, Aramandla Ramesh, Amos Sakwe, Kieosha Williams, Billy R. Ballard, Samuel E. Adunyah. Targeted inhibition of arginase-1 may underline the suppression of the development of colonic neoplasia in a transgenic APC Min mouse model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3710. doi:10.1158/1538-7445.AM2013-3710

Colorectal cancer is a contributing factor for significant mortalities in the United States, with 50,000 deaths per year and around 140,000 new cases expected to be diagnosed in this year. Epidemiological studies have pointed out that obese people have a higher risk for colon cancer. It is well known that overweight or obesity is induced by excess energy intake from dietary fat. Additionally, consumption of well-done red meat and saturated fats, rich in food-borne environmental toxicants such as polycyclic aromatic hydrocarbons (PAHs) has also been implicated as one of the causative factors for sporadic colon cancer. Thus, on one hand, diet-induced obesity and on the other hand exposures to environmental carcinogens contribute to the development of colon cancer. Therefore, the objective of this study was to investigate whether the colon polyp burden was accelerated by diet-induced obesity when exposed to environmental carcinogens.To test this concept, we have employed an adult male transgenic rat model, the Polyposis In the Rat Colon (PIRC) kindred type. This rat is a mutagen-induced nonsense allele of the rat Apc gene on an inbred F344/NTac (F344) genetic background. This rat model is advantageous because of its longer life span and lack of the tumor suppressor gene Apc. We have explored whether tumor burden in PIRC male rat (PIRC-M Heterozygous F344/NTac-Apcam1137) was influenced by the ingestion of different types of fat containing benzo(a)pyrene [B(a)P], a prototypical PAH compound. Treatment consisted of 25 and 50 μg B(a)P/kg body wt., dissolved in tricaprylin, administered to 7-week-old male PIRC rat daily via oral gavage for 60 days. One group of rats received the AIN-76A control diet and the other group, the Western diet throughout the duration of this study. At the end of exposure, rats were sacrificed; colons were retrieved and preserved in 10% formalin for observation of gross pathological changes. An increased prevalence of adenomas in colon of rats that were maintained on Western diet compared to AIN-76A diet and controls (P < 0.05) was noticed. Interestingly, we have also observed adenomas with high grade dysplasia in B(a)P + Western diet group and these incidences were of frequent occurrence at 50 μg/kg compared to 25 μg/kg B(a)P dose group. On the other hand, the B(a)P alone, and AIN-76A diet groups did not show significant differences in the numbers of adenomas and invasive tumors in colon. Also, B(a)P treatment-related changes were seen in body weight and food consumption of rats administered with B(a)P, with the changes more pronounced in the body weight gain of Western diet group compared to the AIN-76A diet group and controls (p < 0.005). In summary, our studies established that Western diet potentiates the development of colon tumors caused by B(a)P in the PIRC rat. Citation Format: Kelly L. Harris, Mohammad S. Niaz, Awadh A. Binhazim, Mary K. Washington, Samuel E. Adunyah, Aramandla Ramesh. Obesity enhances benzo(a)pyrene-induced colon tumorigenesis in a PIRC rat model of colon cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1278. doi:10.1158/1538-7445.AM2013-1278

Sodium butyrate (NaB) is a sodium salt of butyric acid. It is a HDAC1 inhibitor implicated in many studies as a potential therapy for various forms of cancer. Due to its ability to promote both total and gamma globin during erythroid differentiation, low concentrations (1.5 mM) have been shown to activate apoptosis in several cancer cell lines including prostate, endometrial, breast and leukemia. Low concentrations of NaB influence multiple signaling pathways that are known mediators of cytokine production. However, it is not known whether high concentrations alter these pathways having the capacity to modulate cytokine expression in cancer cells and alter the cancer microenvironment, which is the purpose of this study. We exposed U937 leukemia cells to NaB at 5 or 10 mM doses for various time points. The culture media of the cells were harvested and analyzed by Human Multi-Analyte Cytokine ELISArray for levels of cytokines and chemotactic properties via migration assay. As evidence of apoptosis, we monitored cells for caspase-3 activation and cell viability. Our results indicate NaB induces differential expression of several inflammatory cytokines within 24 hours. Significant increases were seen in IL-5, -10, and -17A and a significant decrease for TNF-α. In monocytes, the magnitude of cytokine induction is less as compared to the leukemia cells. Moreover, monocyte migration decreased in a concentration-dependent manner to U937 cells’ exposure to NaB. Also, NaB induced at least 2-fold activation of caspase-3 and reduced cell viability by 60%. To elucidate the mechanism of cytokine expression, activation of AKT and p38 MAPK proteins were investigated using 5 mM NaB. Analyses indicate an increase in phosphorylation of p38, but a decrease in AKT activation over a 24-hour time course. This data suggests while promoting apoptosis, NaB has the potential to influence cancer microenvironment by inducing differential expression of cytokines potentially via the AKT and/or p38 MAPK pathways. This effect may influence the biology of normal and cancer cells altering the status of the patient receiving NaB therapy. *Ms Stephanie Pulliam is supported by NHLBI T32 Pre-doctoral training grant # 2 T32 HL007735-16 to Dr. Adunyah and the CTSA award No. UL1TR000445 from the National Center for Advancing Translational Sciences. Additionally, Dr. Adunyah is supported by NCI Cancer Partnership U54 grant # 5 U54 CA163069-02 and NIMHD MeTRC grant # 8 U54 MD007593-04. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. Citation Format: Stephanie R. Pulliam, Roland Cooper, Samuel E. Adunyah. Regulation of AKT and p38 MAPK by high dose butyrate may influence inflammatory cytokine expression in human U937 leukemia cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4715. doi:10.1158/1538-7445.AM2013-4715

Ovarian cancer, one of inflammation-associated cancers, is the fifth leading cause of cancer deaths among women. Inflammation in the tumor microenvironment is associated with peritoneal tumor dissemination and massive ascites, which contribute to high mortality in ovarian cancer. Tumor suppressor p53 is frequently deleted or mutated in aggressive and high-grade ovarian cancer, probably aggravating cancer progression and increasing mortality. We therefore investigated the influence of p53 on proinflammatory chemokines in ovarian cancer cells. A PCR array of the chemokine network revealed that ovarian cancer cells with low or mutated p53 expression expressed high levels of proinflammatory chemokines such as CXCL1, 2, 3 and 8. Transient transfection of p53 into p53-null ovarian cancer cells downregulated proinflammatory chemokines induced by tumor necrosis factor-α (TNF), a proinflammatory cytokine abundantly expressed in ovarian cancer. Furthermore, p53 restoration or stabilization blocked TNF-induced NF-κB promoter activity and reduced TNF-activated IκB. Restoration of p53 increased ubiquitination of IκB, resulting from concurrently reduced proteasome activity followed by stability of IκB. A ubiquitination PCR array on restoration of p53 did not reveal any significant change in expression except for Mdm2, indicating that the balance between p53 and Mdm2 is more important in regulating NF-κB signaling rather than the direct effect of p53 on ubiquitin-related genes or IκB kinases. In addition, nutlin-3, a specific inducer of p53 stabilization, inhibited proinflammatory chemokines by reducing TNF-activated IκB through p53 stabilization. Taken together, these results suggest that p53 inhibits proinflammatory chemokines in ovarian cancer cells by reducing proteasomal degradation of IκB. Thus, frequent loss or mutation of p53 may promote tumor progression by enhancing inflammation in the tumor microenvironment.

Colorectal carcinoma (CRC) is a serious complication of inflammatory bowel disease (IBD) and accounts for approximately 15% of all IBD-associated deaths. The likelihood of IBD-related carcinoma is greater than that of sporadic CRC. Over one half are diagnosed at stage III or IV when cancer cells have already invaded surrounding tissues and most conventional therapeutics are limited in their success. Therefore the early detection of cancer, which is difficult in IBD, is crucial for its ultimate control and prevention. While mining the colonic mucosal and submucosal layers for biomarkers that differentiate ulcerative colitis (UC) from Crohn's colitis (CC) using Matrix-assisted laser desorption/ionization (MALDI) profiling, we found a signal at m/z 5045 to be more intense in UC. Liquid chromatography–mass spectrometry (LC-MS/MS) analysis allowed identification of this signal as triply charged hemoglobin alpha chain. Macrophages are highly versatile phagocytes active in multiple roles in the immune system and key players in the inflammatory response. Their presence within the inflammatory microenvironment, in some cases, has been proven to increase transformation, angiogenesis, and immunosuppression. In hemorrhagic situations (as in UC), macrophages engulf erythrocytes that are outside the vascular bed and as a result free hemoglobin is released. Hemoglobin induces DNA damage in human colonic cells and is genotoxic. The potential carcinogenic effects of hemoglobin were documented when it was shown that hemoglobin increases the number of aberrant crypt foci in colon mucosa. In the colon, free hemoglobin is expected to increase the production of reactive oxygen (O2) species (ROS) from peroxides via the Fenton reaction, which may be the cause of cellular toxicity and eventually pro-mutagenic lesions. Intracellular reactions with active O2 can result in the initiation and progression of carcinogenesis by induction of gene mutations, chromosomal damage and cytotoxic effects. We hypothesize that elevated expression of mucosal free hemoglobin would be associated with an increased risk of CRC. To validate this hypothesis will require investigating whether hemoglobin could be classified as a proliferative or transforming agent for colon cancer cells by causing reactive oxygen species release and subsequent DNA damage. For this purpose, we will assess the cellular viability of normal colonic cell-lines, NCM 356 and NCM 460. These cell-lines will be treated with hemoglobin at different concentrations to determine the changes in levels of ROS. ROS production will be measured using C-400 staining assay and further analysis will be carried out using FACS. Additionally, we will also examine the potential cytotoxicity of hemoglobin. Supported: MMC-VICC Cancer Partnership Grant#: 3U54CA091408-09S 1; MeTRC grant#: 5U54RR026140-03, and Vanderbilt SPORE in GI Cancer Grant#: P50CA095103.

Colorectal carcinoma (CRC) is a serious complication of inflammatory bowel disease (IBD) and accounts for approximately 15% of all IBD-associated deaths. The likelihood of IBD-related carcinoma is greater than that of sporadic CRC. Over one half are diagnosed at stage III or IV. During the last few years we have worked on mining colon mucosal and submucosal layers for discovery of biomarkers that differentiate ulcerative colitis (UC) from Crohn's colitis (CC). Using LC-MS/MS, we examined signals found to be significantly different between CC and UC samples. We found a signal at m/z 5045 which was more intense in UC samples. The MALDI spectrum did not identify an intact protein entity but did identify hemoglobin chains. Macrophages are highly versatile phagocytes active in multiple roles in the immune system and key players in the inflammatory response. The microenvironment of most inflammation is filled with a large population of macrophages. In IBD, studies have found that macrophages can count for more than 50% of the exudative mass. Their presence within the inflammatory microenvironment, in some cases, has been proven to increase transformation, angiogenesis, and immunosuppression. In hemorrhagic situations (as in UC) macrophages engulf erythrocytes and as a result release free heme iron (heFe). Earlier studies observed that heFe has cellular proliferation effects on colon cancer cells. Recently, the potential carcinogenic effects of heFe were documented when it was shown that heFe increases the number of aberrant crypt foci in colon mucosa. In the colon, iron is expected to increase the production of reactive oxygen (O2) species (ROS) from peroxides via the Fenton reaction, which may be the cause of cellular toxicity and even pro-mutagenic lesions. Intracellular reactions with active O2 can result in the initiation and progression of carcinogenesis by induction of gene mutations, chromosomal damage and cytotoxic effects. We hypothesize that elevated expression of mucosal free heFe would be associated with an increased risk of UC-associated CRC. To validate this will require investigating whether hemoglobin could be classified as a proliferative or transforming agent for colon cancer cells by causing reactive oxygen species release. For this purpose, we plan to study the cellular viability of differentiated colon cell line (cancer: CCL 224, CCL 227 and normal: NCM 356 and NCM 460) after administration of hemoglobin at different concentrations. ROS production will be investigated in each step. Additionally, we intend to examine the potential cytotoxicity of hemoglobin. Supported: MMC-VICC Cancer Partnership Grant # 3U54CA091408-09S 1 (SEA & HLM); MeTRC grant # 5U54RR026140-03 (SEA), and Vanderbilt SPORE in GI Cancer Grant # P50CA095103 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2058. doi:1538-7445.AM2012-2058

Statistics released by the American Cancer society indicates that colon cancer is the third leading cause of cancer cases and related deaths in America. In addition to dietary preferences and lifestyle habits, exposure to toxicants such as benzo(a)pyrene (BaP) is one of the major contributing factors to the development of sporadic colon cancer. Our laboratory has validated the ApcMin mouse model to study BaP-induced colon carcinogenesis. Ongoing studies in our laboratory are focused towards elucidating the anticarcinogenic effects of resveratrol (RVT) against colon tumors. Our studies thus far have shown a decrease in the incidence, size, and number of adenomas formed in the colon of mice exposed to BaP and RVT, compared to BaP exposure alone. Since biotransformation of toxicants is one of the key steps for initiating carcinogenesis, the objective of this study was to investigate whether RVT exposure simultaneously or prior to BaP treatment alters BaP biotransformation and bioavailability in ApcMin mice. The BaP treatment consisted of BaP-only administration (in peanut oil) at a dose of 100 μg/kg body weight (bw) via oral gavage over a 60 day period (group I); BaP (100 μg/kg bw) co-administered with RVT (in 10% ethanol + 90% deionized water) at a dose of 45 μg/kg bw (group II); RVT administered for 1 week prior to BaP dosing (group III). Blood, colon and liver samples were collected at the end of exposure period. The expression of BaP biotransformation enzymes (CYP1A1, CYP1B1 and GST) in liver and colon were assayed at the level of protein and enzyme activities. Plasma, liver and colon tissue samples were analyzed by reverse phase-HPLC for BaP metabolites. Resveratrol exposure both prior to- and concurrent with BaP exposure caused a decrease in the expression and activity of CYP1A1/1B1 enzymes and an increase in GST enzymes both in liver and colon. Additionally, our studies revealed a decrease in concentrations of organic (Phase I) metabolites in plasma, liver and colon in mice that received RVT + BaP compared to mice that received RVT alone. In contrast, an opposite trend was noted with aqueous (Phase II) metabolites registering an increase in mice that received RVT + BaP compared to mice that received RVT alone. Between the two RVT-treatment strategies, concurrent administration of RVT appeared to limit BaP bioactivation compared to RVT treatment prior to BaP exposure. In summary, our results suggest that RVT provides a preventive effect against BaP-induced colon cancer initiation and progression in ApcMin mice (funded by NIH grants 5T32HL007735-12, 1F31ES019432-01A1 and 5RO1CA142845-O2). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5459. doi:1538-7445.AM2012-5459

Purpose: Differentiating ulcerative colitis (UC) and Crohn's colitis (CC) may be inaccurate in up to 30% of inflammatory bowel disease (IBD) cases due to overlapping features of these colitides. IBD-associated colorectal cancer (CRC) is frequently diagnosed in an advanced stage. Mass-spectrometric (MS) proteomic patterns found in colonic submucosa have been shown to discriminate UC and CC. To verify this, we aimed to conduct pilot gene expression analyses via microarray assessment of colonic submucosa. The significances of these genes to CRC initiation is as yet not been elucidated. Methods: Laser capture microdissection of colonic submucosa from UC (n=8), CC (n=8), and normal (NL, n=8) samples was performed. The submucosal mRNA was extracted using the PicoPure(TM) RNA Isolation Kit. Comprehensive gene expression analysis of the pooled mRNA from each group was then performed using the Affymetrix GeneChip® Gene 1.0 ST Array System. To detect changes for UC, we compared UC to CC and UC to NL; and for CC, we compared CC to UC and CC to NL. Results: 28,869 genes were represented on the array by 26 probes spread across the full length of each gene. When comparing UC to CC, 138 genes showed at least a 5-fold significant overexpression (p Conclusions: Microarray assessments show significantly different gene overexpression in UC and CC colonic submucosa, with a few select genes showing dramatic overexpression. Preliminary results indicate that we have the tools in hand to successfully validate the results from the microarray analysis using TR-qPCR and immunohistochemistry (IHC). These genes may assist in delineation of the previously identified differentiating MS submucosal proteomic peaks, potentially facilitating protein biomarker identification to discriminate the inflammatory colitides. These genes may relate to CRC initiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-450. doi:10.1158/1538-7445.AM2011-LB-450

Purpose: Differentiating Crohn's colitis (CC) and ulcerative colitis (UC) can be challenging even in combination of clinical, endoscopic, radiologic and histopathology examination. Biomarker studies have thus far been unsuccessful for disease delineation. We aim to use unique tissue proteomic methods to evaluate colonic tissue layers for potential biomarkers to identify CC vs. UC. Methods: Fresh-frozen colon specimens from resections for IBD and/or colorectal cancer were retrospectively retrieved. Colitis diagnoses were histologically re-confirmed by a blinded gastrointestinal pathologist. Three sample groups (n=5 each group) were examined: normal colon from CRC specimens (control), UC & CC. MALDI-MS was used to profile mucosal and submucosal compartments individually. Frozen tissues were sectioned at ∼10–15 μm for mounting onto either metal or conductive glass target plates (the glass plates allowing for histologic and MALDI-MS analysis on the same section). Sinapinic acid (20 mg/mL in 50:50 acetonitrile: 0.1% TFA in water) was used to give the best combination of uniform crystal coverage and signal quality for direct tissue protein analysis. Results: MALDI-MS achieved high mass accuracy (±0.01 Daltons) in the lower mass range ( Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-463. doi:10.1158/1538-7445.AM2011-LB-463

Purpose: Ulcerative colitis (UC)-associated colon cancer is frequently diagnosed in an advanced stage. SAA1 is a secreted protein made in the liver and circulates in low levels in blood. SAA1 gene is expressed in colon carcinomas and is a chemoattractant with induction of migration, adhesion, and tissue infiltration of monocytes and Polymorphnuclear-leucocytes (PMN-L). Methods: Laser capture microdissection of colonic submucosa from UC (n=8), Crohn's colitis (CC) (n=8)), and normal (NL, n=8) samples was performed. The submucosal mRNA was extracted using the PicoPure(TM) RNA Isolation Kit. Comprehensive gene expression analysis of the pooled mRNA from each group was then performed using the Affymetrix GeneChip® Gene 1.0 ST Array System. Statistical comparisons of UC vs. CC and NL as well as CC versus UC and NL were performed (Wilcoxon Rank Test). To detect changes for UC, we compared UC to CC and UC to NL; and for CC, we compared CC to UC and CC to NL. Results: Analysis of all UC vs. CC and NL controls showed 28,869 genes which were represented on the array by 26 probes spread across the full length of each gene. When compared UC vs. CC and NL SAA1 remained the most upregulated gene showing a 56.8-folds and 91.3-folds (p< 0.0001) over expression. Conclusion: Microarray assessments show significantly overexpression of SAA1 in UC specimens. SAA1 has a role in local inflammation in the microenvironment of malignant tissue and is expressed in colon carcinomas. Our finding may potentially facilitate and validate the SAA1 as a new early predictive clinical marker and a unique target for designing novel selective inhibitors for therapeutic intervention of UC-associated CRC. Acknowledgement: 3U54CA091408–09S1 (MMC-VICC partnership) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-428. doi:10.1158/1538-7445.AM2011-LB-428

Sodium butyrate (NaB) is a sodium salt of butyric acid. It is a strong HDAC1 inhibitor, which has been implicated in many studies as a potential therapy for various forms of cancer including prostate cancer. Also, it has been suggested as a potential therapy for hemoglobin disorders including sickle cell diseases and leukemia because of its ability to promote both total globin and gamma globin synthesis during erythroid differentiation. Previously NaB has been shown to active apoptosis in several cancer cell lines including U937 and K562 leukemia cell lines. However, it is not known whether it has the capability to modulate cytokines expression in cancer cells and alter the cancer microenvironment, which was the purpose of this study. We exposed U937 leukemia cells to NaB at 5 mM or 10 mM for 24 or 48 hours, conditions under which NaB is known to induce apoptosis in these cells. The culture media of the cells were harvested and analyzed by Human Multi-Analyte Cytokine ELISArray method and by protein/antibody cytokine arrays for levels of TH17-type and Inflammatory cytokines. We also monitored cells for caspase 3 activation and cell viability as evidence of apoptosis. Our results indicate that NaB induces differential expression of several TH17-type and Inflammatory cytokines from 2 to 10-fold within 24 hours. The ranking order of induction of TH17-type cytokines by NaB was IL-17A>IL-5>IL-4>G-CSF>IL-10>TNF>IL-12>IFNγ while induction of inflammatory cytokines was of the ranking order: IL-1A>IL-8>IL-10. Also within 24 hours, NaB treatment caused at least 2-fold activation of caspase 3 and reduced the cell viability by 60 %. These observations indicate that NaB induces differential expression of both TH17 and Inflammatory cytokines during induction of apoptosis. The data also suggest that while promoting cancer cell death by apoptosis, NaB also has the potential to influence cancer microenvironment by inducing differential expression and secretion of cytokines. Such an effect could influence the biology of both the normal and cancer cells and could alter the status of the patient who is receiving NaB therapy. Further studies are in progress to determine whether NaB has similar effects in human monocytes. Supported by NCI U54 cancer grants # 5 U54 CA091408-09. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3831.