Sinicrope, Frank A.

Abstract Purpose: Celecoxib has been shown to induce cell death in conjunction with its chemopreventive effects. However, the mechanisms by which celecoxib induces cell death are incompletely understood. We determined whether celecoxib can induce autophagy, a process of cell destruction whereby cytoplasmic proteins and organelles are sequestered in vacuoles and delivered to lysosomes for degradation and recycling. Both apoptosis and autophagy have been shown to be negatively regulated by pro-survival Bcl-2 proteins, and the small molecule Bcl-2/Bcl-xL antagonist (ABT-737), can enhance both processes. Since autophagy can be pro-survival or pro-death depending upon the cellular context, we determined whether autophagy inhibition can drive colon cancer cells into apoptosis. Methods: HCT116 (wild-type and Bax−/−), HT29, SW480 colorectal cancer cell lines were treated with celecoxib (0–120 µM) and/or ABT-737 (Abbott; 0–5 µM) in the presence or absence of a pan caspase inhibitor, z-VAD-fmk. Cell viability was determined using the MTS assay and apoptosis was analyzed by Annexin V FITC labeling. Immunoblotting was used to analyze caspase cleavage and LC3 conversion. GFP-LC3B cellular localization was determined by confocol microscopy. Autophagic flux was determined by LC3 immunoblotting in the presence of the lysosome inhibitor, bafilomycin A1. Knockdown of Bcl-xL, Vps34 or Atg8/LC3B were achieved by lentiviral shRNA or siRNA. Results: Celecoxib was shown to induce apoptosis that was attenuated by ectopic Bcl-2 or Bax knockout. ABT-737 synergistically enhanced celecoxib-induced apoptosis as shown by caspase cleavage and Annexin V labeling. Celecoxib also induced autophagy, as shown by conversion of the autophagosome-associated protein light chain 3 (LC3) from a cytosolic (LC3I) to a membrane-bound (LC3II) form as shown by immunoblotting, and a punctate fluorescence pattern of ectopic GFP-LC3 protein. Celecoxib-induced conversion of LC3 that was due to autophagy induction, as shown using the lysosome inhibitor, Bafilomycin A1 that produced an accumulation of LC3II. Furthermore, ABT-737 enhanced LC3 conversion by celecoxib. Autophagy inhibition was achieved using 3-methyladenine (3-MA), wortmannin or knockdown of Vps34 or Atg8/LC3B using siRNA. Inhibition of autophagy enhanced apoptosis as shown by caspase cleavage and Bid truncation in cells treated with celecoxib plus ABT-737. Furthermore, the addition of 3-MA to celecoxib plus ABT-737 produced a 5-fold increase in apoptosis that was associated with caspase cleavage and cytochrome c release. Conclusions: Celecoxib induces both apoptosis and autophagy that can both be potentiated by ABT-737. Inhibition of autophagy was shown to potently drive cells into apoptosis, suggesting a novel therapeutic strategy against colon cancer cells. Citation Information: Cancer Prev Res 2010;3(1 Suppl):B46.

Abstract Purpose: Obesity is associated with an increased risk of colon cancer. However, the influence of body mass index (BMI) upon the outcome of colon cancer survivors and its relationship to gender remains unknown. Experimental Design: BMI (kg/m2) was calculated and categorized in patients with TNM stage II and III colon carcinomas (n= 4,381) enrolled in 7 randomized trials of 5-fluorouracil-based adjuvant chemotherapy. Cox proportional hazards modeling was used to determine the association of BMI with disease-free and overall survival. Results: Among colon cancer patients, 868 (20%) were obese (BMI ≥30 kg/m2) of which 606 were class 1 (BMI 30–34 kg/m2) and 262 were class 2,3 (BMI ≥35 kg/m2). Obese versus normal-weight patients were more likely to be younger, have distal tumors, show intact DNA mismatch repair (MMR), and have increased lymph node metastases (p <0.017). The association of BMI and MMR status was dependent upon patient age (p= 0.031). In a multivariate analysis, BMI was significantly associated with both disease-free (p=0.030) and overall survival (p=0.0017). Men with class 2,3 obesity showed reduced overall survival compared to normal weight men [HR 1.35 (1.02, 1.79); p=0.039]. Women with class I obesity had reduced overall survival [HR 1.24 (1.01, 1.53), p=0.045] compared to normal weight women. BMI was not predictive of therapeutic benefit. Conclusions: Obesity is associated with intact DNA mismatch repair status and was an adverse, independent prognostic variable in colon cancer survivors. Interventions to reduce obesity have the potential to improve patient outcomes in colon cancer survivors. Citation Information: Cancer Prev Res 2010;3(1 Suppl):B111.

Abstract Background: Increasing colorectal cancer screening (CRCS) is important for attaining the Healthy People 2020 goal of reducing CRC-related morbidity and mortality. Evaluating CRCS trends can help identify shifts in CRCS, and specific groups that might be targeted for CRCS. Methods: We utilized medical records to describe population-based adherence to average-risk CRCS guidelines from 1997 to 2008 in Olmsted County, MN. CRCS trends were analyzed overall and by gender, age, and adherence to screening mammography (women only). We also carried out an analysis to examine whether CRCS is being initiated at the recommended age of 50. Results: From 1997 to 2008, the size of the total eligible sample ranged from 20,585 to 21,468 people. CRCS increased from 22% to 65% for women and from 17% to 59% for men (P < 0.001 for both) between 1997 and 2008. CRCS among women current with mammography screening increased from 26% to 74%, and this group was more likely to be adherent to CRCS than all other subgroups analyzed (P < 0.001).The mean ages of screening initiation were stable throughout the study period, with a mean age of 55 years among both men and women in 2008. Conclusion: Although overall CRCS tripled during the study period, there is still room for improvement. Impact: Working to decrease the age at first screening, exploration of gender differences in screening behavior, and targeting women adherent to mammography but not to CRCS seem warranted. Cancer Epidemiol Biomarkers Prev; 21(2); 347–50. ©2011 AACR.

Abstract Purpose: We examined the frequency, tumor characteristics, and prognostic impact of HER2 protein expression and gene amplification in patients with curatively resected esophageal adenocarcinoma (EAC). Experimental Design: HER2 expression was analyzed by immunohistochemistry (IHC) in surgical EAC specimens (n = 713). Gene amplification was examined by FISH in a large subset (n = 344). Most tumors were T3–4 (66%) or node positive (72%); 95% were located in the esophagus or gastroesophageal junction. No patient received neoadjuvant therapy. Cox models were used. Results: Overall, 17% of EACs were HER2 positive (i.e., IHC3+ or IHC2+ with amplification), with strong agreement between HER2 amplification (HER2/CEP17 ratio ≥2) and expression (κ = 0.83). HER2 positivity was significantly associated with lower tumor grade, less invasiveness, fewer malignant nodes, and the presence of adjacent Barrett's esophagus (BE). EACs with BE had higher odds of HER2 positivity than EACs without BE, independent of pathologic features [OR = 1.8 (95% CI: 1.1–2.8), P = 0.014]. Among all cases, HER2 positivity was significantly associated with disease-specific survival (DSS) in a manner that differed by the presence or absence of BE (Pinteraction = 0.0047). In EACs with BE, HER2 positivity was significantly associated with improved DSS [HR = 0.54 (95% CI: 0.35–0.84), P = 0.0065] and overall survival (P = 0.0022) independent of pathologic features, but was not prognostic among EACs without BE. Conclusions: HER2 positivity was shown in 17% of resected EACs and associated with reduced tumor aggressiveness. EACs with BE had nearly twice the odds of being HER2 positive and, within this subgroup, HER2 positivity was independently associated with improved survival. Clin Cancer Res; 18(2); 546–54. ©2012 AACR.

PurposeAlthough the importance of obesity in colon cancer risk and outcome is recognized, the association of body mass index (BMI) with DNA mismatch repair (MMR) status is unknown.Patients and MethodsBMI (kg/m2) was determined in patients with TNM stage II or III colon carcinomas (n = 2,693) who participated in randomized trials of adjuvant chemotherapy. The association of BMI with MMR status and survival was analyzed by logistic regression and Cox models, respectively.ResultsOverall, 427 (16%) tumors showed deficient MMR (dMMR), and 630 patients (23%) were obese (BMI ≥ 30 kg/m2). Obesity was significantly associated with younger age (P = .021), distal tumor site (P = .012), and a lower rate of dMMR tumors (10% v 17%; P < .001) compared with normal weight. Obesity remained associated with lower rates of dMMR (odds ratio, 0.57; 95% CI, 0.41 to 0.79; P < .001) after adjusting for tumor site, stage, sex, and age. Among obese patients, rates of dMMR were lower in men compared with women (8% v 13%; P = .041). Obesity was associated with higher recurrence rates (P = .0034) and independently predicted worse disease-free survival (DFS; hazard ratio [HR], 1.37; 95% CI, 1.14 to 1.64; P = .0010) and overall survival (OS), whereas dMMR predicted better DFS (HR, 0.59; 95% CI, 0.47 to 0.74; P < .001) and OS. The favorable prognosis of dMMR was maintained in obese patients.ConclusionColon cancers from obese patients are less likely to show dMMR, suggesting obesity-related differences in the pathogenesis of colon cancer. Although obesity was independently associated with adverse outcome, the favorable prognostic impact of dMMR was maintained among obese patients.

Purpose Given that smoking affects body mass index (BMI) and survival, stratification by smoking status may be required to determine the true prognostic impact of BMI. Although obesity increases risk for developing esophageal adenocarcinoma (EAC), the prognostic influence of obesity and its potential modification by smoking status is unknown in this disease. Patients and Methods All patients (N = 778) underwent potentially curative esophagectomy. BMI was calculated using measured height and weight at surgery and categorized as obese (≥ 30 kg/m 2 ), overweight (25 to 29.9 kg/m 2 ), or normal (18.5 to 24.9 kg/m 2 ). Cigarette smoking was categorized as never or ever. The association of BMI with disease-specific survival (DSS), disease-free survival (DFS), and overall survival (OS) was determined by Cox regression. Results Excess BMI was significantly associated with DSS in a manner that differed substantially by smoking status (P for interaction = .023). Among never smokers, obesity was significantly associated with adverse DSS (hazard ratio [HR] = 2.11; 95% CI, 1.31 to 3.43; P = .002), DFS (HR = 2.03; 95% CI, 1.30 to 3.18; P = .002), and OS (HR = 1.97; 95% CI, 1.24 to 3.14; P = .004), as compared with normal weight, after adjusting for covariates. By contrast, among ever smokers, obesity was not prognostic, and overweight status was significantly associated with favorable survival in univariate, but not multivariate, analysis. Conclusion Obesity among never smokers was independently associated with two-fold worsening of DSS, DFS, and OS after surgery for EAC, after adjusting for known prognostic factors. These data, in one of the largest reported resected EAC cohorts, are the first to show an adverse prognostic impact of obesity in EAC.

Abstract Autophagy is a homeostatic, catabolic degradation process whereby cellular proteins and organelles are engulfed by autophagosomes, digested in lysosomes, and recycled to sustain cellular metabolism. Autophagy has dual roles in cancer, acting as both a tumor suppressor by preventing the accumulation of damaged proteins and organelles and as a mechanism of cell survival that can promote the growth of established tumors. Tumor cells activate autophagy in response to cellular stress and/or increased metabolic demands related to rapid cell proliferation. Autophagy-related stress tolerance can enable cell survival by maintaining energy production that can lead to tumor growth and therapeutic resistance. As shown in preclinical models, inhibition of autophagy restored chemosensitivity and enhanced tumor cell death. These results established autophagy as a therapeutic target and led to multiple early phase clinical trials in humans to evaluate autophagy inhibition using hydroxychloroquine in combination with chemotherapy or targeted agents. Targeting autophagy in cancer will provide new opportunities for drug development, because more potent and specific inhibitors of autophagy are needed. The role of autophagy and its regulation in cancer cells continues to emerge, and studies aim to define optimal strategies to modulate autophagy for therapeutic advantage. Mol Cancer Ther; 10(9); 1533–41. ©2011 AACR.

Abstract Patients with Barrett's esophagus (BE) and dysplasia are candidates for chemopreventive strategies to reduce cancer risk. We determined the effects of difluoromethylornithine (DMFO) on mucosal polyamines, gene expression, and histopathology in BE. Ten patients with BE and low-grade dysplasia participated in a single-arm study of DFMO (0.5 g/m2/d) given continuously for 6 months. Esophagoscopy with biopsies was conducted at baseline, 3, 6, and 12 months. Dysplasia was graded by a gastrointestinal pathologist. Audiology was assessed (at baseline and at 6 months). Mucosal polyamines were measured by high-performance liquid chromatography. Microarray-based gene expression was analyzed using a cDNA two-color chip. DFMO suppressed levels of the polyamines putrescine (P = 0.02) and spermidine (P = 0.02) and the spermidine/spermine ratio (P &lt; 0.01) in dysplastic BE (6 months vs. baseline) that persisted at 6 months following drug cessation. Among the top 25 modulated genes, we found those regulating p53-mediated cell signaling (RPL11), cell-cycle regulation (cyclin E2), and cell adhesion and invasion (Plexin1). DFMO downregulated Krüppel-like factor 5 (KLF5), a transcription factor promoting cell proliferation, and suppressed RFC5 whose protein interacts with proliferating cell nuclear antigen. Histopathology showed regression of dysplasia (n = 1), stable disease (n = 8), and progression to high-grade dysplasia (n = 1). Polyamines were suppressed in the responder to a greater extent than in stable cases. DFMO was well tolerated, and one patient had subclinical, unilateral ototoxicity. DFMO suppressed mucosal polyamines and modulated genes that may be mechanistically related to its chemopreventive effect. Further study of DFMO for the chemoprevention of esophageal cancer in BE patients is warranted. Cancer Prev Res; 4(6); 829–39. ©2011 AACR.

Abstract Purpose: Obesity is associated with an increased risk of colon cancer. However, the influence of body mass index (BMI) upon the prognosis of colon cancer survivors and its relationship to gender remains unknown. Experimental Design: BMI (kg/m2) was categorized in patients with TNM stage II and III colon carcinomas (n= 4,381) enrolled in 7 randomized trials of 5-fluorouracil-based adjuvant chemotherapy. Cox proportional hazards models were used to determine the association of BMI with disease-free (DFS) and overall survival (OS). Results: Among colon cancer patients, 868 (20%) were obese (BMI &gt;30 kg/m2) of which 606 were class 1 (BMI 30-34 kg/m2) and 262 were class 2,3 (BMI &gt;35 kg/m2). Obese versus normal-weight patients were more likely to be younger (p=0.038), have distal tumors (0.058), show intact DNA mismatch repair (MMR) (p=0.035), and have more lymph node metastases (p=0.017). In a multivariate analysis, BMI was significantly associated with both DFS (p=0.030) and OS (p=0.0017). Men with class 2,3 obesity showed reduced OS compared to normal weight men [HR 1.35 (1.02, 1.79); p=0.039]. Women with class I obesity had reduced OS [HR 1.24 (1.01, 1.53), p=0.045] compared to normal weight women. Overweight status was associated with improved OS in men (p= 0.006) and underweight women had significantly worse OS (p= 0.019). BMI was not predictive of therapeutic benefit. Conclusions: Obesity is an independent prognostic variable in colon cancer survivors and shows gender-related differences. These data suggest that obesity-related biological factors can influence clinical outcome. 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 904.