Laura Biddick

Eflornithine (DFMO) Prevents Progression of Pancreatic Cancer by Modulating Ornithine Decarboxylase Signaling

Ornithine decarboxylase (ODC) is the key rate-limiting enzyme in the polyamine synthesis pathway and it is overexpressed in a variety of cancers. We found that polyamine synthesis and modulation of ODC signaling occurs at early stages of pancreatic precursor lesions and increases as the tumor progresses in Kras-activated p48Cre/+-LSL-KrasG12D/+ mice. Interest in use of the ODC inhibitor eflornithine (DFMO) as a cancer chemopreventive agent has increased in recent years since ODC was shown to be transactivated by the c-myc oncogene and to cooperate with the ras oncogene in malignant transformation of epithelial tissues. We tested the effects of DFMO on pancreatic intraepithelial neoplasias (PanIN) and their progression to pancreatic ductal adenocarcinoma (PDAC) in genetically engineered Kras mice. The KrasG12D/+ mice fed DFMO at 0.1% and 0.2% in the diet showed a significant inhibition (P < 0.0001) of PDAC incidence compared with mice fed control diet. Pancreatic tumor weights were decreased by 31% to 43% (P < 0.03–0.001) with both doses of DFMO. DFMO at 0.1% and 0.2% caused a significant suppression (27% and 31%; P < 0.02–0.004) of PanIN 3 lesions (carcinoma in situ). DFMO-treated pancreas exhibited modulated ODC pathway components along with decreased proliferation and increased expression of p21/p27 as compared with pancreatic tissues derived from mice fed control diet. In summary, our preclinical data indicate that DFMO has potential for chemoprevention of pancreatic cancer and should be evaluated in other PDAC models and in combination with other drugs in anticipation of future clinical trials. Cancer Prev Res; 7(12); 1198–209. ©2014 AACR.

Chemoprevention of Urothelial Cell Carcinoma Growth and Invasion by the Dual COX–LOX Inhibitor Licofelone in UPII-SV40T Transgenic Mice

Epidemiologic and clinical data suggest that use of anti-inflammatory agents is associated with reduced risk for bladder cancer. We determined the chemopreventive efficacy of licofelone, a dual COX–lipoxygenase (LOX) inhibitor, in a transgenic UPII-SV40T mouse model of urothelial transitional cell carcinoma (TCC). After genotyping, six-week-old UPII-SV40T mice (n = 30/group) were fed control (AIN-76A) or experimental diets containing 150 or 300 ppm licofelone for 34 weeks. At 40 weeks of age, all mice were euthanized, and urinary bladders were collected to determine urothelial tumor weights and to evaluate histopathology. Results showed that bladders of the transgenic mice fed control diet weighed 3 to 5-fold more than did those of the wild-type mice due to urothelial tumor growth. However, treatment of transgenic mice with licofelone led to a significant, dose-dependent inhibition of the urothelial tumor growth (by 68.6%–80.2%, P < 0.0001 in males; by 36.9%–55.3%, P < 0.0001 in females) compared with the control group. The licofelone diet led to the development of significantly fewer invasive tumors in these transgenic mice. Urothelial tumor progression to invasive TCC was inhibited in both male (up to 50%; P < 0.01) and female mice (41%–44%; P < 0.003). Urothelial tumors of the licofelone-fed mice showed an increase in apoptosis (p53, p21, Bax, and caspase3) with a decrease in proliferation, inflammation, and angiogenesis markers (proliferating cell nuclear antigen, COX-2, 5-LOX, prostaglandin E synthase 1, FLAP, and VEGF). These results suggest that licofelone can serve as potential chemopreventive for bladder TCC. Cancer Prev Res; 7(7); 708–16. ©2014 AACR.

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

High number of regulatory T cells (Tregs), both circulating and at the tumor site, often indicates a poor prognosis in CRC patients possibly impairing natural killer (NK) cell function. To determine the role of Tregs in CRC development and their effects on NK cells, we created novel transgenic Rag‐Apc mice that lack T cells and develop spontaneous intestinal tumors, and we adoptively transferred Tregs or transiently depleted NK cells during initial stages of tumorigenesis. In 6‐weeks old Rag‐Apc mice containing microscopic intestinal tumors adoptive transfer of Tregs or transient NK cell depletion dramatically associated with an increase in intestinal tumor multiplicity and tumor size, with significantly decreased survival rates. Importantly, Treg transfer increased small intestinal polyp formation up to 65% (P < 0.0005) and increased colon tumors multiplicities by 84% (P < 0.0001) with a significant decrease in NK cells as compared to control mice. Similarly, in NK depleted mice, colon tumor multiplicities increased up to 40% and small intestinal polyp formation up to 60% (P < 0.0001). Treg transfer or NK cell transient depletion markedly increased interleukin (IL)‐22 systemically and the inflammatory signaling molecules P2X7R, and STAT3 in the tumors; and impaired production of the tumor suppressor interferon (IFN)‐γ systemically. Notably, IL‐22 binding protein (IL‐22 BP) was associated with NKs and a significant decrease was seen at the tumor site in mice adoptively transferred with Tregs or depleted of NK cells. Our results suggest that adoptive transfer of Tregs aggressively promote intestinal tumorigenesis by decreasing NK cell number and activity by modulating IL‐22 BP.

Systemic Chromosome Instability Resulted in Colonic Transcriptomic Changes in Metabolic, Proliferation, and Stem Cell Regulators in Sgo1−/+ Mice

Colon cancer is the second most lethal cancer and is predicted to claim 49,700 lives in the United States this year. Chromosome instability (CIN) is observed in 80% to 90% of colon cancers and is thought to contribute to colon cancer progression and recurrence. To investigate the impact of CIN on colon cancer development, we developed shugoshin-1 (Sgo1) haploinsufficient (-/+) mice, an animal model focusing on mitotic error-induced CIN. In this study, we analyzed signature changes in the colonic transcriptome of Sgo1(-/+) mice to examine the molecular events underlying the altered carcinogenesis profiles in Sgo1(-/+) mice. We performed next-generation sequencing of normal-looking colonic mucosal tissue from mice treated with the carcinogen azoxymethane after 24 weeks. Transcriptome profiling revealed 349 hits with a 2-fold expression difference threshold (217 upregulated genes, 132 downregulated genes, P < 0.05). Pathway analyses indicated that the Sgo1-CIN tissues upregulated pathways known to be activated in colon cancer, including lipid metabolism (z score 4.47), Notch signaling (4.47), insulin signaling (3.81), and PPAR pathways (3.75), and downregulated pathways involved in immune responses including allograft rejection (6.69) and graft-versus-host disease (6.54). Notably, stem cell markers were also misregulated. Collectively, our findings demonstrate that systemic CIN results in transcriptomic changes in metabolism, proliferation, cell fate, and immune responses in the colon, which may foster a microenvironment amenable to cancer development. Therefore, therapeutic approaches focusing on these identified pathways may be valuable for colon cancer prevention and treatment.

Antagonizing pathways leading to differential dynamics in colon carcinogenesis in Shugoshin1 (Sgo1)-haploinsufficient chromosome instability model

Colon cancer is the second most lethal cancer. It is predicted to claim 50,310 lives in 2014. Chromosome Instability (CIN) is observed in 80–90% of colon cancers, and is thought to contribute to colon cancer progression and recurrence. However, there are no animal models of CIN that have been validated for studies of colon cancer development or drug testing. In this study, we sought to validate a mitotic error‐induced CIN model mouse, the Shugoshin1 (Sgo1) haploinsufficient mouse, as a colon cancer study model. Wild‐type and Sgo1−/+ mice were treated with the colonic carcinogen, azoxymethane (AOM). We tracked colon tumor development 12, 24, and 36 wk after treatment to assess progression of colon tumorigenesis. Initially, more precancerous lesions, Aberrant Crypt Foci (ACF), developed in Sgo1−/+ mice. However, the ACF did not develop straightforwardly into larger tumors. At the 36‐wk endpoint, the number of gross tumors in Sgo1−/+ mice was no different from that in wild‐type controls. However, Copy Number Variation (CNV) analysis indicated that fully developed colon tumor in Sgo1−/+ mice carried 13.75 times more CNV. Immunohistological analyses indicated that Sgo1−/+ mice differentially expressed IL‐6, Bcl2, and p16INK4A. We propose that formation of ACF in Sgo1−/+ mice is facilitated by the IL6‐STAT3‐SOCS3 oncogenic pathway and by the Bcl2‐anti‐apoptotic pathway, yet further development of the ACF to tumors is inhibited by the p16INK4A tumor suppressor pathway. Manipulating these pathways would be beneficial for inhibiting development of colon cancer with CIN.

Small-Molecule Inhibition of GCNT3 Disrupts Mucin Biosynthesis and Malignant Cellular Behaviors in Pancreatic Cancer.

Pancreatic cancer is an aggressive neoplasm with almost uniform lethality and a 5-year survival rate of 7%. Several overexpressed mucins that impede drug delivery to pancreatic tumors have been therapeutically targeted, but enzymes involved in mucin biosynthesis have yet to be preclinically evaluated as potential targets. We used survival data from human patients with pancreatic cancer, next-generation sequencing of genetically engineered Kras-driven mouse pancreatic tumors and human pancreatic cancer cells to identify the novel core mucin-synthesizing enzyme GCNT3 (core 2 β-1,6 N-acetylglucosaminyltransferase). In mouse pancreatic cancer tumors, GCNT3 upregulation (103-fold; P < 0.0001) was correlated with increased expression of mucins (5 to 87-fold; P < 0.04-0.0003). Aberrant GCNT3 expression was also associated with increased mucin production, aggressive tumorigenesis, and reduced patient survival, and CRISPR-mediated knockout of GCNT3 in pancreatic cancer cells reduced proliferation and spheroid formation. Using in silico small molecular docking simulation approaches, we identified talniflumate as a novel inhibitor that selectively binds to GCNT3. In particular, docking predictions suggested that three notable hydrogen bonds between talniflumate and GCNT3 contribute to a docking affinity of -8.3 kcal/mol. Furthermore, talniflumate alone and in combination with low-dose gefitinib reduced GCNT3 expression, leading to the disrupted production of mucins in vivo and in vitro Collectively, our findings suggest that targeting mucin biosynthesis through GCNT3 may improve drug responsiveness, warranting further development and investigation in preclinical models of pancreatic tumorigenesis. Cancer Res; 76(7); 1965-74. ©2016 AACR.

Targeting mTOR and p53 Signaling Inhibits Muscle Invasive Bladder Cancer In Vivo

Urothelial tumors, accompanied by mutations of the tumor suppressor protein TP53 and dysregulation of mTOR signaling, are frequently associated with aggressive growth and invasiveness. We investigated whether targeting these two pathways would inhibit urothelial tumor growth and progression. Six-week-old transgenic UPII-SV40T male mice (n = 15/group) were fed control diet (AIN-76A) or experimental diets containing mTOR inhibitor (rapamycin, 8 or 16 ppm), p53 stabilizing agent [CP31398 (CP), 150 ppm], or a combination. Mice were euthanized at 40 weeks of age. Urinary bladders were collected and evaluated to determine tumor weight and histopathology. Each agent alone, and in combination, significantly inhibited tumor growth. Treatment with rapamycin alone decreased tumor weight up to 67% (P < 0.0001). Similarly, CP showed approximately 77% (P < 0.0001) suppression of tumor weight. The combination of low-dose rapamycin and CP led to approximately 83% (P < 0.0001) inhibition of tumor weight. There was no significant difference in tumor weights between rapamycin and CP treatments (P > 0.05). However, there was a significant difference between 8 ppm rapamycin and the combination treatment. Tumor invasion was also significantly inhibited in 53% (P < 0.005) and 66% (P < 0.0005) mice after 8 ppm and 16 ppm rapamycin, respectively. However, tumor invasion was suppressed in 73% (P < 0.0001) mice when CP was combined with 8 ppm rapamycin. These results suggest that targeting two or more pathways achieve better treatment efficacy than a single-agent high-dose strategy that could increase the risk of side effects. A combination of CP and rapamycin may be a promising method of inhibiting muscle-invasive urothelial transitional cell carcinoma. Cancer Prev Res; 9(1); 53–62. ©2015 AACR.

Abstract 1005: Targeting COX-LOX and EGFR pathways simultaneously by licofelone and gefitinib lead to complete blockade of progression of PanINs to pancreatic ductal adenocarcinoma

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Pancreatic ductal adenocarcinoma (PDAC), a devastating disease with dismal prognosis (5-year survival rate of <5%), is the fourth leading cause of cancer deaths in the USA. Developing novel strategies to prevent or delay progression of pancreatic cancer is currently of intense interest. Clinically, COX-2 (93%), 5-LOX (90%) and EGRF (87%) are over-expressed in human PDAC. COX-2 & 5-LOX metabolites play a pivotal role in cell signaling and proliferation, and their release has been shown in response to EGF and growth stimuli. Both COX-2 & EGFR are known to synergistically activate oncogenic signaling. To target COX-2, 5-LOX and EGFR simultaneously, we tested the effects of licofelone, a novel dual 5-LOX-COX-2 inhibitor and gefitinib, an EGFR inhibitor individually and in combination on pancreatic intraepithelial neoplasms (PanINs) and their progression to PDAC in p48Cre/+-LSL-KrasG12D/+ mice. Six-week old male and female KrasG12D/+ (∼35/group) mice were fed (AIN-76A) diets containing 0, 250 ppm licofelone, 100 ppm gefitinib or combination of both for 38 weeks. Pancreata were collected, weighed, and evaluated histopathologically for PanINs and PDAC. To understand molecular mechanisms, we analyzed levels of proliferation, apoptosis, cell cycle and modification in cancer stem cell makers (DCAMKL-1 & CD133); COX-2, 5-LOX, PCNA, p21, p2X7, cyclin D1, β-catenin, EGFR, Cav-1, p38, C2GNT and mucin expressions by Radiomatic HPLC, IHC, IHF, Tunel, Western blotting, and/or RT-PCR methods. Results suggest that control diet fed mice showed 64 and 82% incidence of PDAC in female and male mice, respectively. Dietary licofelone and gefitinib significantly inhibited incidence of PDAC in both male (72 & 90%, respectively, p<0.0001) and female (90 & 85%, respectively, p<0.0001) mice. Most importantly, the combination drug treatment showed complete (100%, p<0.0001) inhibition of PDAC incidence in both genders of mice. Also, a significant suppression of PanIN 3 (56, 50 and 70% P<0.001) was observed in mice fed by licofelone, gefitinib and their combination, respectively. The pancreas of mice fed combination diets showed significantly reduced cancer stem-cells positive for DCAMKL-1 and CD133 (P<0.0001) and a significant inhibition of COX-2, 5-LOX, PCNA, cyclin D1, EGFR, p38, Cav-1 and β-catenin expression levels (p<0.05-0.0002); and increased apoptosis, when compared to the pancreatic cancer derived from control diet or individual drug fed mice. Importantly, mucin synthesis and C2GNT were significantly inhibited by gefitinib and combination but not licofelone, suggesting that EGFR activation is linked to pancreatic ductal mucin biosynthesis. In summary, targeting the 5-LOX/COX-2 and EGFR pathways simultaneously may provide synergistic and/or additive chemopreventive effects in complete suppression of PDAC. {Supported by NCI-CN-N01-53300}. 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 1005. doi:1538-7445.AM2012-1005

Abstract 2969: Combinational Targeting of EGFR and ODC pathways by Gefitinib and DFMO lead to complete blockade of PanIN progression to pancreatic ductal adenocarcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Pancreatic cancer (PC) is a devastating disease with almost uniform lethality despite aggressive treatment (5-year survival rate of <6%). Developing novel strategies to prevent/delay/inhibit progression of PC is currently of intense interest. Epidermal growth factor receptor (EGFR) is over-expressed in ∼87% human pancreatic cancers and plays a pivotal role in tumor cell proliferation. An interaction between ornithine decarboxylase (ODC) overexpression and EGFR was suggested by the elevation of EGFR Tyr-K activity. Clinical and preclinical studies have clearly demonstrated chemopreventive potential of difloromethyl ornithine (DFMO), an ODC inhibitor. Higher doses of gefitinib (EGFR inhibitor) and DFMO has been associated with skin- and oto-toxicity respectively. Hence, to target EGFR and ODC simultaneously, we tested the effects of lower doses of gefitinib and DFMO individually and in combination on pancreatic intraepithelial neoplasms (PanINs) and their progression to pancreatic ductal adenocarcinoma (PDAC) in p48Cre/+-LSL-KrasG12D/+ transgenic mice. Six-week old male and female KrasG12D/+ (24-34/group) mice were fed (AIN-76A) diets containing 0%, 0.01% gefitinib, 0.1% DFMO or combination of both for 38 weeks. Pancreata were collected, weighed, and evaluated histopathologically for PanINs and PDAC. To understand molecular mechanisms, we analyzed levels of proliferation, apoptosis and cell cycle makers; PCNA, p21, β-catenin, Cav-1, Bcl-XL, c-MYC, cyclin E and pERK expressions by IHC, IHF, Western blotting, and/or RT-PCR methods. Results suggest that control diet fed mice showed 80 and 65% incidence of PDAC in male and female mice, respectively. Dietary gefitinib and DFMO significantly inhibited incidence of PDAC in both male (90 & 87%, respectively, p<0.0001) and female (84 & 75%, respectively, p<0.0001) mice. Most importantly, the combination drug treatment showed complete (100%, p<0.0001) inhibition of PDAC incidence in both genders of mice. Also, significant suppression of PanIN 3 (carcinoma in-situ) was observed in mice fed by gefitinib, DFMO and their combination. Importantly, ∼77% of the pancreas was free from lesions and carcinoma in the combination treatment compared to only 4 % in control and 39 and 23 % in gefitinib and DFMO fed mice respectively. The pancreas of mice fed combination diets showed a significant inhibition of PCNA, β-catenin, Cav-1, Bcl-XL, c-MYC, cyclin E and pERK expression levels (p<0.05-0.001); and increased p21 when compared to the pancreatic cancer derived from control diet or individual drug fed mice. In summary, targeting the EGFR and ODC pathways simultaneously may provide synergistic and/or additive chemopreventive effects in suppression of PC and has significant potential for undertaking clinical trials of pancreatic cancer chemoprevention. {Supported by NCI-CN-N01-53300}. Citation Format: Altaf Mohammed, Naveena B. Janakiram, Rebekah L. Ritchie, Laura Biddick, Misty Brewer, Stan Lightfoot, Vernon E. Steele, Chinthalapally V. Rao. Combinational Targeting of EGFR and ODC pathways by Gefitinib and DFMO lead to complete blockade of PanIN progression to pancreatic ductal adenocarcinoma. [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 2969. doi:10.1158/1538-7445.AM2014-2969

Abstract B111: Early and Delayed Rapamycin prevents NNK-induced lung adenocarcinoma in A/J mice

Lung cancer is the leading cause of cancer deaths. Several studies have shown that nicotine and its metabolite, the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), activate the Akt/mTOR pathway, which maintains lung tumor cell proliferation and survival. Targeting this pathway might be one of the options for lung cancer chemoprevention. We studied chemopreventive effects of Rapamycin by intervening at two different stages of NNK-induced lung adenoma and adenocarcinoma formation in female A/J mice. At 6 weeks of age, groups of mice were randomized and fed control AIN-76A modified diet. At 7 weeks of age, 40 mice from each group intended for carcinogen treatment received one dose of 10 μmol NNK /mouse by i.p. injection. Three weeks after the NNK treatment, groups of mice (25/group) were fed either control or experimental diets containing 8 or 16 ppm Rapamycin as an early stage intervention. Mice were continued on the respective diets and killed at 20 weeks (10 mice/group) after NNK-treatment and tumors were evaluated by histopathological methods. At 20 weeks - after tumors were allowed to form, another two groups (15 mice/group) were administered experimental diets containing 8 or 16 ppm Rapamycin as a late stage intervention and continued on the respective diets. Mice were killed at 36 weeks (15 mice/group) after both early stage and late stage interventions. Administration of 8 or 16 ppm Rapamycin as an early or a late stage intervention significantly suppressed lung adenoma and adenocarcinoma formation (p<0.01-0.0001) at 20 weeks and at 36 weeks. Rapamycin caused suppression of NNK-induced lung adenocarcinoma at both stages, but the effect was more pronounced at the early stage intervention >50-60% (P< 0.0001). Rapamycin treatment decreased the size of NNK-induced tumors from >2.10 to <∼0.75 mm3 (p=0.0056). Lung tumors harvested from mice exposed to Rapamycin showed a significant decrease in phosphorylation of S6 kinase (p-S6K) and phospho-Akt (p-Akt) when compared to control in both the early and the late stage intervention studies. Immunohistochmical analysis revealed that lung tumors from Rapamycin-treated mice had significantly reduced proliferating cell nuclear antigen (P<0.05) and p-S6 (P<0.005)-positive cells. These observations support the benefit of targeting the Akt/mTOR pathways in development of early interventions for lung cancer chemoprevention. {Supported by NIH, NCI grants NO1-CN-53300} Citation Format: Jagan Mohan Reddy Patlolla, Levy Kopelovich, Li Qian, Laura Biddick, Yuting Zhang, Dhimant Desai, Shantu Amin, Stan Lightfoot, Chinthalapally V. Rao. Early and delayed rapamycin prevents NNK-induced lung adenocarcinoma in A/J mice. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B111.