Proanthocyanidins mitigate bile acid-induced changes in GSTT2 levels in a panel of racially diverse patient-derived primary esophageal cell cultures.

Persistent and symptomatic reflux of gastric and duodenal contents, known as gastroesophageal reflux disease (GERD), is the strongest risk factor for esophageal adenocarcinoma (EAC). Despite similar rates of GERD and other risk factors across racial groups, EAC progression disproportionately impacts Caucasians. We recently reported that elevated tissue levels of the detoxification enzyme GSTT2 in the esophagi of Blacks compared to Caucasians may contribute protection. Herein, we extend our research to investigate whether cranberry proanthocyanidins (C-PAC) mitigate bile acid-induced damage and GSTT2 levels utilizing a racially diverse panel of patient-derived primary esophageal cultures. We have shown that C-PACs mitigate reflux-induced DNA damage through GSTT2 upregulation in a rat esophageal reflux model, but whether effects are recapitulated in humans or differentially based on race remains unknown. We isolated normal primary esophageal cells from Black and Caucasian patients and assessed GSTT2 protein levels and cellular viability following exposure to a bile acid cocktail with and without C-PAC treatment. Constitutive GSTT2 levels were significantly elevated in Black (2.9-fold) compared to Caucasian patients, as were GSTT2 levels in Black patients with GERD. C-PAC treatment induced GSTT2 levels 1.6-fold in primary normal esophageal cells. GSTT2 induction by C-PAC was greatest in cells with constitutively low GSTT2 expression. Overall, C-PAC mitigated bile-induced reductions of GSTT2 and subsequent loss of cell viability regardless of basal GSTT2 expression or race. These data support that C-PAC may be a safe efficacious agent to promote epithelial fitness through GSTT2 induction and in turn protect against bile acid-induced esophageal injury.

Constitutively Higher Level of GSTT2 in Esophageal Tissues From African Americans Protects Cells Against DNA Damage.

BACKGROUND & AIMS: African American and European American individuals have a similar prevalence of gastroesophageal reflux disease (GERD), yet esophageal adenocarcinoma (EAC) disproportionately affects European American individuals. We investigated whether the esophageal squamous mucosa of African American individuals has features that protect against GERD-induced damage, compared with European American individuals. METHODS: We performed transcriptional profile analysis of esophageal squamous mucosa tissues from 20 African American and 20 European American individuals (24 with no disease and 16 with Barrett's esophagus and/or EAC). We confirmed our findings in a cohort of 56 patients and analyzed DNA samples from patients to identify associated variants. Observations were validated using matched genomic sequence and expression data from lymphoblasts from the 1000 Genomes Project. A panel of esophageal samples from African American and European American subjects was used to confirm allele-related differences in protein levels. The esophageal squamous-derived cell line Het-1A and a rat esophagogastroduodenal anastomosis model for reflux-generated esophageal damage were used to investigate the effects of the DNA-damaging agent cumene-hydroperoxide (cum-OOH) and a chemopreventive cranberry proanthocyanidin (C-PAC) extract, respectively, on levels of protein and messenger RNA (mRNA). RESULTS: We found significantly higher levels of glutathione S-transferase theta 2 (GSTT2) mRNA in squamous mucosa from African American compared with European American individuals and associated these with variants within the GSTT2 locus in African American individuals. We confirmed that 2 previously identified genomic variants at the GSTT2 locus, a 37-kb deletion and a 17-bp promoter duplication, reduce expression of GSTT2 in tissues from European American individuals. The nonduplicated 17-bp promoter was more common in tissue samples from populations of African descendant. GSTT2 protected Het-1A esophageal squamous cells from cum-OOH-induced DNA damage. Addition of C-PAC increased GSTT2 expression in Het-1A cells incubated with cum-OOH and in rats with reflux-induced esophageal damage. C-PAC also reduced levels of DNA damage in reflux-exposed rat esophagi, as observed by reduced levels of phospho-H2A histone family member X. CONCLUSIONS: We found GSTT2 to protect esophageal squamous cells against DNA damage from genotoxic stress and that GSTT2 expression can be induced by C-PAC. Increased levels of GSTT2 in esophageal tissues of African American individuals might protect them from GERD-induced damage and contribute to the low incidence of EAC in this population.

Expression, modulation, and clinical correlates of the autophagy protein Beclin-1 in esophageal adenocarcinoma.

Esophageal adenocarcinoma (EAC) is characterized by rapidly increasing incidence and mortality rates and poor survival. Efficacious preventive and treatment options are urgently needed. An increasing number of pharmacologic agents targeting cancer cell death via autophagy mechanisms are being evaluated in hopes of circumventing apoptotic and therapeutic resistance. We report for the first time, loss of Beclin-1, a key mediator of autophagy, was significantly linked to prognostic factors in EAC. Specifically, Beclin-1 expression loss occurred in 49.0% of EAC patients versus 4.8% of controls. There was a significant inverse correlation between loss of Beclin-1 with histologic grade and tumor stage supporting a tumor suppressive role for Beclin-1. Autophagy modulation linked to cell death was examined in EAC cell lines following treatment with a proanthocyanidin-rich cranberry extract, C-PAC, and the commonly used autophagy inducer, rapamycin. C-PAC induced Beclin-1-independent autophagy in EAC cells characterized by reduced phosphorylation at serine 15 and 93, and significant cell death induction. In contrast, rapamycin-induced autophagy resulted in concomitant, increases in total Beclin-1 levels as well as Beclin-1-phosphorylation in a cell line specific manner, leading to long-term cell survival. Furthermore, autophagic LC3-II was induced by C-PAC following siRNA suppression of Beclin-1 in EAC cells. Together these data support a prognostic role of Beclin-1 in EAC with evidence that Beclin-dependent autophagy induction is agent specific. Future studies are necessary to fully interrogate the role autophagy plays in the progression of normal tissue to EAC and how specific agents targeting autophagic mechanisms can be efficaciously applied for cancer prevention or treatment. © 2015 Wiley Periodicals, Inc.

Prebiotic proanthocyanidins inhibit bile reflux-induced esophageal adenocarcinoma through reshaping the gut microbiome and esophageal metabolome.

The gut and local esophageal microbiome progressively shift from healthy commensal bacteria to inflammation-linked pathogenic bacteria in patients with gastroesophageal reflux disease, Barrett's esophagus, and esophageal adenocarcinoma (EAC). However, mechanisms by which microbial communities and metabolites contribute to reflux-driven EAC remain incompletely understood and challenging to target. Herein, we utilized a rat reflux-induced EAC model to investigate targeting the gut microbiome-esophageal metabolome axis with cranberry proanthocyanidins (C-PAC) to inhibit EAC progression. Sprague-Dawley rats, with or without reflux induction, received water or C-PAC ad libitum (700 µg/rat/day) for 25 or 40 weeks. C-PAC exerted prebiotic activity abrogating reflux-induced dysbiosis and mitigating bile acid metabolism and transport, culminating in significant inhibition of EAC through TLR/NF-κB/TP53 signaling cascades. At the species level, C-PAC mitigated reflux-induced pathogenic bacteria (Streptococcus parasanguinis, Escherichia coli, and Proteus mirabilis). C-PAC specifically reversed reflux-induced bacterial, inflammatory, and immune-implicated proteins and genes, including Ccl4, Cd14, Crp, Cxcl1, Il6, Il1b, Lbp, Lcn2, Myd88, Nfkb1, Tlr2, and Tlr4, aligning with changes in human EAC progression, as confirmed through public databases. C-PAC is a safe, promising dietary constituent that may be utilized alone or potentially as an adjuvant to current therapies to prevent EAC progression through ameliorating reflux-induced dysbiosis, inflammation, and cellular damage.

Cranberry Proanthocyanidins Mitigate Reflux-Induced Transporter Dysregulation in an Esophageal Adenocarcinoma Model.

We recently reported that cranberry proanthocyanidins (C-PACs) inhibit esophageal adenocarcinoma (EAC) by 83% through reversing reflux-induced bacterial, inflammatory and immune-implicated proteins and genes as well as reducing esophageal bile acids, which drive EAC progression. This study investigated whether C-PACs' mitigation of bile reflux-induced transporter dysregulation mechanistically contributes to EAC prevention. RNA was isolated from water-, C-PAC- and reflux-exposed rat esophagi with and without C-PAC treatment. Differential gene expression was determined by means of RNA sequencing and RT-PCR, followed by protein assessments. The literature, coupled with the publicly available Gene Expression Omnibus dataset GSE26886, was used to assess transporter expression levels in normal and EAC patient biopsies for translational relevance. Significant changes in ATP-binding cassette (ABC) transporters implicated in therapeutic resistance in humans (i.e., Abcb1, Abcb4, Abcc1, Abcc3, Abcc4, Abcc6 and Abcc10) and the transport of drugs, xenobiotics, lipids, and bile were altered in the reflux model with C-PACs' mitigating changes. Additionally, C-PACs restored reflux-induced changes in solute carrier (SLC), aquaporin, proton and cation transporters (i.e., Slc2a1, Slc7a11, Slc9a1, Slco2a1 and Atp6v0c). This research supports the suggestion that transporters merit investigation not only for their roles in metabolism and therapeutic resistance, but as targets for cancer prevention and targeting preventive agents in combination with chemotherapeutics.

Role of IL4 and GMCSF in Predicting Survival in Esophageal Cancer.

BACKGROUND: Esophageal cancer (EC) originates in the setting of chronic inflammation. Although previous studies have sought to understand the role of inflammatory signaling in EC, the effect of these immunologic changes on patient outcomes remains understudied. This study's objective was to identify relationships between cytokine levels and prognosis in a mixed cohort of esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC) patients. STUDY DESIGN: A total of 37 serum cytokines were profiled at the time of resection using multiplex ELISA in 47 patients (42 esophageal adenocarcinoma, 5 esophageal squamous cell carcinoma). Cytokine levels were median-binarized and assessed using Cox regression models. Findings were validated at the RNA level using The Cancer Genome Atlas EC cohort (81 esophageal adenocarcinoma, 81 esophageal squamous cell carcinoma). RESULTS: Univariable analysis revealed high serum interleukin 4 (IL4) and granulocyte-macrophage colony-stimulating factor (GMCSF) were negatively associated with overall survival (p = 0.046, p = 0.040). Multivariable analysis determined both high serum IL4 or high serum GMCSF were negatively associated with survival independent of important clinical factors (hazard ratio [HR] 7.55, p < 0.001; HR 5.24, p = 0.001). These findings were validated at the RNA level in The Cancer Genome Atlas EC cohort, where multivariable analysis identified high IL4 expression, high CSF2 expression (encodes GMCSF), and advanced pathologic stage as independent negative predictors of survival when controlled for clinical factors (HR 2.35, p = 0.012; HR 1.97, p = 0.040). CONCLUSIONS: These results show that high IL4/GMCSF levels are negatively associated with survival in EC. These relationships are independent of pathologic stage and are identified across modalities, histologic subtypes, and the presence/absence of neoadjuvant therapy.

Prebiotic proanthocyanidins inhibit bile reflux-induced esophageal adenocarcinoma through reshaping the gut microbiome and esophageal metabolome.

The gut and local esophageal microbiome progressively shift from healthy commensal bacteria to inflammatory-linked pathogenic bacteria in patients with gastroesophageal reflux disease, Barrett's esophagus and esophageal adenocarcinoma (EAC). However, mechanisms by which microbial communities and metabolites contribute to reflux-driven EAC remain incompletely understood and challenging to target. Herein, we utilized a rat reflux-induced EAC model to investigate targeting the gut microbiome-esophageal metabolome axis with cranberry proanthocyanidins (C-PAC) to inhibit EAC progression. Sprague Dawley rats, with or without reflux-induction received water or C-PAC ad libitum (700 µg/rat/day) for 25 or 40 weeks. C-PAC exerted prebiotic activity abrogating reflux-induced dysbiosis, and mitigating bile acid metabolism and transport, culminating in significant inhibition of EAC through TLR/NF-κB/P53 signaling cascades. At the species level, C-PAC mitigated reflux-induced pathogenic bacteria (Clostridium perfringens, Escherichia coli, and Proteus mirabilis). C-PAC specifically reversed reflux-induced bacterial, inflammatory and immune-implicated proteins and genes including Ccl4, Cd14, Crp, Cxcl1, Il6, Il1ß, Lbp, Lcn2, Myd88, Nfkb1, Tlr2 and Tlr4 aligning with changes in human EAC progression, as confirmed through public databases. C-PAC is a safe promising dietary constituent that may be utilized alone or potentially as an adjuvant to current therapies to prevent EAC progression through ameliorating reflux-induced dysbiosis, inflammation and cellular damage.

Cranberry Polyphenols in Esophageal Cancer Inhibition: New Insights.

Esophageal adenocarcinoma (EAC) is a cancer characterized by rapidly rising incidence and poor survival, resulting in the need for new prevention and treatment options. We utilized two cranberry polyphenol extracts, one proanthocyanidin enriched (C-PAC) and a combination of anthocyanins, flavonoids, and glycosides (AFG) to assess inhibitory mechanisms utilizing premalignant Barrett's esophagus (BE) and EAC derived cell lines. We employed reverse phase protein arrays (RPPA) and Western blots to examine cancer-associated pathways and specific signaling cascades modulated by C-PAC or AFG. Viability results show that C-PAC is more potent than AFG at inducing cell death in BE and EAC cell lines. Based on the RPPA results, C-PAC significantly modulated 37 and 69 proteins in JH-EsoAd1 (JHAD1) and OE19 EAC cells, respectively. AFG treatment significantly altered 49 proteins in both JHAD1 and OE19 cells. Bioinformatic analysis of RPPA results revealed many previously unidentified pathways as modulated by cranberry polyphenols including NOTCH signaling, immune response, and epithelial to mesenchymal transition. Collectively, these results provide new insight regarding mechanisms by which cranberry polyphenols exert cancer inhibitory effects targeting EAC, with implications for potential use of cranberry constituents as cancer preventive agents.

Characterizing isoform switching events in esophageal adenocarcinoma.

Isoform switching events with predicted functional consequences are common in many cancers, but characterization of switching events in esophageal adenocarcinoma (EAC) is lacking. Next-generation sequencing was used to detect levels of RNA transcripts and identify specific isoforms in treatment-naïve esophageal tissues ranging from premalignant Barrett's esophagus (BE), BE with low- or high-grade dysplasia (BE.LGD, BE.HGD), and EAC. Samples were stratified by histopathology and TP53 mutation status, identifying significant isoform switching events with predicted functional consequences. Comparing BE.LGD with BE.HGD, a histopathology linked to cancer progression, isoform switching events were identified in 75 genes including KRAS, RNF128, and WRAP53. Stratification based on TP53 status increased the number of significant isoform switches to 135, suggesting switching events affect cellular functions based on TP53 mutation and tissue histopathology. Analysis of isoforms agnostic, exclusive, and shared with mutant TP53 revealed unique signatures including demethylation, lipid and retinoic acid metabolism, and glucuronidation, respectively. Nearly half of isoform switching events were identified without significant gene-level expression changes. Importantly, two TP53-interacting isoforms, RNF128 and WRAP53, were significantly linked to patient survival. Thus, analysis of isoform switching events may provide new insight for the identification of prognostic markers and inform new potential therapeutic targets for EAC.

Genetic polymorphisms of multiple DNA repair pathways impact age at diagnosis and TP53 mutations in breast cancer.

Defective DNA repair may contribute to early age and late stage at time of diagnosis and mutations in critical tumor suppressor genes, such as TP53 in breast cancer. Using DNA samples from 436 breast cancer cases (374 Caucasians and 62 African-Americans), we tested these associations with 18 non-synonymous single-nucleotide polymorphisms (nsSNPs) in four DNA repair pathways: (i) base excision repair: ADPRT V762A, APE1 D148E, XRCC1 R194W/R280H/R399Q and POLD1 R119H; (ii) double-strand break repair: NBS1 E185Q and XRCC3 T241M; (iii) mismatch repair: MLH1 I219V, MSH3 R940Q/T1036A and MSH6 G39E and (iv) nucleotide excision repair: ERCC2 D312N/K751Q, ERCC4 R415Q, ERCC5 D1104H and XPC A499V/K939Q. Younger age at diagnosis (<50) was associated with ERCC2 312 DN/NN genotypes [odds ratio (OR) = 1.76; 95% confidence interval (CI) = 1.10, 2.81] and NBS1 185 QQ genotype (OR = 3.09; 95% CI = 1.47, 6.49). The XPC 939 QQ genotype was associated with TP53 mutations (OR = 5.80; 95% CI = 2.23, 15.09). There was a significant trend associating younger age at diagnosis (<50) with increasing numbers of risk genotypes for ERCC2 312 DN/NN, MSH6 39 EE and NBS1 185 QQ (P(trend) < 0.001). A similar significant trend was also observed associating TP53 mutations with increasing numbers of risk genotypes for XRCC1 399 QQ, XPC 939 QQ, ERCC4 415 QQ and XPC 499 AA (P(trend) < 0.001). Our pilot data suggest that nsSNPs of multiple DNA repair pathways are associated with younger age at diagnosis and TP53 mutations in breast cancer and larger studies are warranted to further evaluate these associations.

MicroRNA alterations in Barrett's esophagus, esophageal adenocarcinoma, and esophageal adenocarcinoma cell lines following cranberry extract treatment: Insights for chemoprevention.

BACKGROUND: Aberrant expression of small noncoding endogenous RNA molecules known as microRNAs (miRNAs) is documented to occur in multiple cancer types including esophageal adencarcinoma (EAC) and its only known precursor, Barrett's esophagus (BE). Recent studies have linked dysregulation of specific miRNAs to histological grade, neoplastic progression and metastatic potential. MATERIALS AND METHODS: Herein, we present a summary of previously reported dysregulated miRNAs in BE and EAC tissues as well as EAC cell lines and evaluate a cranberry proanthocyanidin rich extract's (C-PAC) ability to modulate miRNA expression patterns of three human EAC cell lines (JHEso-Ad-1, OE33 and OE19). RESULTS: A review of 13 published studies revealed dysregulation of 87 miRNAs in BE and EAC tissues, whereas 52 miRNAs have been reported to be altered in BE or EAC cell lines, with 48% overlap with miRNA changes reported in tissues. We report for the first time C-PAC-induced modulation of five miRNAs in three EAC cell lines resulting in 26 validated gene targets and identification of key signaling pathways including p53, angiogenesis, T-cell activation and apoptosis. Additionally, mutiple cancer related networks were ideintified as modulated by C-PAC utilizing Kyoto Encyclopedia of Genes and Genomes (KEGG), Protein Analysis Through Evolutionary Relationships (PANTHER), and MetaCore analysis tools. CONCLUSIONS: Study results support the cancer inhibitory potential of C-PAC is in part attributable to C-PAC's ability to modify miRNA profiles within EAC cells. A number of C-PAC-modulated miRNAs have been been identified as dysregulated in BE and EAC. Further insights into miRNA dysregulation and modulation by select cancer preventive agents will support improved targeted interventions in high-risk cohorts.

Cranberry proanthocyanidins mediate growth arrest of lung cancer cells through modulation of gene expression and rapid induction of apoptosis.

Cranberries are rich in bioactive constituents purported to enhance immune function, improve urinary tract health, reduce cardiovascular disease and more recently, inhibit cancer in preclinical models. However, identification of the cranberry constituents with the strongest cancer inhibitory potential and the mechanism associated with cancer inhibition by cranberries remains to be elucidated. This study investigated the ability of a proanthocyanidin rich cranberry fraction (PAC) to alter gene expression, induce apoptosis and impact the cell cycle machinery of human NCI-H460 lung cancer cells. Lung cancer is the leading cause of cancer-related deaths in the United States and five year survival rates remain poor at 16%. Thus, assessing potential inhibitors of lung cancer-linked signaling pathways is an active area of investigation.

Immune determinants of Barrett's progression to esophageal adenocarcinoma.

Esophageal adenocarcinoma (EAC) develops from Barrett's esophagus (BE), a chronic inflammatory state that can progress through a series of transformative dysplastic states before tumor development. While molecular and genetic changes of EAC tumors have been studied, immune microenvironment changes during Barrett's progression to EAC remain poorly understood. In this study, we identify potential immunologic changes that can occur during BE-to-EAC progression. RNA sequencing (RNA-Seq) analysis on tissue samples from EAC patients undergoing surgical resection demonstrated that a subset of chemokines and cytokines, most notably IL6 and CXCL8, increased during BE progression to EAC. xCell deconvolution analysis investigating immune cell population changes demonstrated that the largest changes in expression during BE progression occurred in M2 macrophages, pro-B cells, and eosinophils. Multiplex immunohistochemical staining of tissue microarrays showed increased immune cell populations during Barrett's progression to high-grade dysplasia. In contrast, EAC tumor sections were relatively immune poor, with a rise in PD-L1 expression and loss of CD8+ T cells. These data demonstrate that the EAC microenvironment is characterized by poor cytotoxic effector cell infiltration and increased immune inhibitory signaling. These findings suggest an immunosuppressive microenvironment, highlighting the need for further studies to explore immune modulatory therapy in EAC.

Disparities in Head and Neck Cancer: A Case for Chemoprevention with Vitamin D.

Blacks experience disproportionate head and neck cancer (HNC) recurrence and mortality compared to Whites. Overall, vitamin D status is inversely associated to HNC pointing to a potential protective linkage. Although hypovitaminosis D in Blacks is well documented it has not been investigated in Black HNC patients. Thus, we conducted a prospective pilot study accessing vitamin D status in newly diagnosed HNC patients stratified by race and conducted in vitro studies to investigate mechanisms associated with potential cancer inhibitory effects of vitamin D. Outcome measures included circulating levels of vitamin D, related nutrients, and risk factor characterization as well as dietary and supplemental estimates. Vitamin D-based in vitro assays utilized proteome and microRNA (miR) profiling. Nineteen patients were enrolled, mean circulating vitamin D levels were significantly reduced in Black compared to White HNC patients, 27.3 and 20.0 ng/mL, respectively. Whites also supplemented vitamin D more frequently than Blacks who had non-significantly higher vitamin D from dietary sources. Vitamin D treatment of HNC cell lines revealed five significantly altered miRs regulating genes targeting multiple pathways in cancer based on enrichment analysis (i.e., negative regulation of cell proliferation, angiogenesis, chemokine, MAPK, and WNT signaling). Vitamin D further altered proteins involved in cancer progression, metastasis and survival supporting a potential role for vitamin D in targeted cancer prevention.

Mitochondria-targeted magnolol inhibits OXPHOS, proliferation, and tumor growth via modulation of energetics and autophagy in melanoma cells.

INTRODUCTION: Melanoma is an aggressive form of skin cancer for which there are no effective drugs for prolonged treatment. The existing kinase inhibitor antiglycolytic drugs (B-Raf serine/threonine kinase or BRAF inhibitors) are effective for a short time followed by a rapid onset of drug resistance. PRESENTATION OF CASE: Here, we show that a mitochondria-targeted analog of magnolol, Mito-magnolol (Mito-MGN), inhibits oxidative phosphorylation (OXPHOS) and proliferation of melanoma cells more potently than untargeted magnolol. Mito-MGN also inhibited tumor growth in murine melanoma xenografts. Mito-MGN decreased mitochondrial membrane potential and modulated energetic and mitophagy signaling proteins. DISCUSSION: Results indicate that Mito-MGN is significantly more potent than the FDA-approved OXPHOS inhibitor in inhibiting proliferation of melanoma cells. CONCLUSION: These findings have implications in the treatment of melanomas with enhanced OXPHOS status due to metabolic reprogramming or drug resistance.

Circular RNA circHIPK3 modulates autophagy via MIR124-3p-STAT3-PRKAA/AMPKα signaling in STK11 mutant lung cancer.

The role of circular RNA in cancer is emerging. A newly reported circular RNA HIPK3 (circHIPK3) is critical in cell proliferation of various cancer types, although its role in non-small cell lung cancer (NSCLC), has yet to be elucidated. Our results provided evidence that silencing of circHIPK3 significantly impaired cell proliferation, migration, invasion and induced macroautophagy/autophagy. Mechanistically, we uncovered that autophagy was induced upon loss of circHIPK3 via the MIR124-3p-STAT3-PRKAA/AMPKa axis in STK11 mutant lung cancer cell lines (A549 and H838). STAT3 abrogation as well as transfection with a MIR124-3p mimic, recapitulated the induction of autophagy. We also demonstrated antagonistic regulation on autophagy between circHIPK3 and linear HIPK3 (linHIPK3). We therefore propose that the ratio between circHIPK3 and linHIPK3 (C:L ratio) may reflect autophagy levels in cancer cells. We observed that a high C:L ratio (>0.49) was an indicator of poor survival, especially in advanced-stage NSCLC patients. These results support that circHIPK3 is a key autophagy regulator in a subset of lung cancer and has potential clinical use as a prognostic factor. The circular RNA HIPK3 (circHIPK3) functions as an oncogene and autophagy regulator may potential use as a prognostic marker and therapeutic target in lung cancer.Abbreviations 3-MA: 3-methyladenine; AMPK: AMP-activated protein kinase; ATG7: autophagy related 7; Baf-A: bafilomycin A1; BECN1: beclin 1; circHIPK3: circular HIPK3; CQ: chloroquine; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; HIPK3: homeodomain interacting protein kinase 3; IL6R: interleukin 6 receptor; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; NSCLC: non-small cell lung cancer; RFP: red fluorescent protein; RPS6KB1/S6K: ribosomal protein S6 kinase B1; SQSTM1/p62: sequestosome 1; STAT3: signal transducer and activator of transcription 3; STK11: serine/threonine kinase 11.

Intake of plant foods and associated nutrients in prostate cancer risk.

Plant foods and associated nutrients may impact prostate cancer (PC) risk and survival. Therefore, we compared dietary intake, mainly plant food groups among 382 controls and 478 PC cases (373 incident and 105 prevalent cases). Caucasian controls had significantly higher daily servings of vegetables (3.4 vs. 2.5, P= 0.002) and fruits and/or fruit juices (1.6 vs. 1.3, P = 0.02) compared to African American controls. In Caucasians, incident cases reported lower intake of fiber, vitamin C, vitamin A, alpha -carotene, beta -carotene, cryptoxanthin, folate, genistein, daidzein, and fruits and/or fruit juice than controls and/or prevalent cases. In African Americans, incident cases had lower intake of alpha -carotene compared to controls and prevalent cases. Reduced PC risk was associated with the highest tertile of cryptoxanthin (OR = 0.51; 95% CI = 0.35-0.75), fiber (OR = 0.56; 95% CI = 0.35-0.89), vitamin C (OR = 0.60; 95% CI = 0.41-0.88), and fruits and/or fruit juices (OR = 0.46; 95% CI = 0.31-0.68), with significant linear trends. Increased risk of PC was associated with the highest tertile of protein (OR = 1.99; 95% CI = 1.05-3.79) and daily servings of grains (OR = 1.99; 95% CI = 1.23-3.22) with significant linear trends. In summary, we demonstrate racial/ethnic differences in dietary intake of plant foods. The significantly higher consumption of protective dietary constituents among prevalent cases compared to incident cases suggests that PC survivors may be amenable to dietary change.

Effects of a topically applied bioadhesive berry gel on loss of heterozygosity indices in premalignant oral lesions.

PURPOSE: The aim of this study was to assess the effects of topical application of a 10% (w/w) freeze-dried black raspberry (FBR) gel on oral intraepithelial neoplasia (IEN) variables that included histologic diagnoses and loss of heterozygosity (LOH) indices. Microsatellite instability and/or LOH at tumor suppressor gene-associated chromosomal loci have been associated with a higher risk for oral IEN progression to oral squamous cell carcinoma. Previously, our laboratories have shown that FBRs are well tolerated and possess potent antioxidant, apoptotic, and differentiation-inducing properties. EXPERIMENTAL DESIGN: Each participant with IEN served as their own internal control. Before treatment, all lesions were photographed, and lesional tissue was hemisected to obtain a pretreatment diagnosis and baseline biochemical and molecular variables. Gel dosing (0.5 g applied four times daily for 6 weeks) was initiated 1 week after the initial biopsy. Genomic DNA was isolated from laser-captured basilar and suprabasilar surface epithelial cells followed by PCR amplification using primer sets that targeted known and presumed tumor suppressor gene loci associated with INK4a/ARF, p53, and FHIT. Allelic imbalance was determined by sequence analysis using normal participant tissues to establish microsatellite marker peak patterns and allele sizes. RESULTS: Confirming earlier phase I data, none of the 27 participants developed FBR gel-associated toxicities. Furthermore, our results show histologic regression in a subset of patients as well as statistically significant reduction in LOH at tumor suppressor gene-associated loci. CONCLUSIONS: These preliminary data suggest that further evaluation of berry gels for oral IEN chemoprevention is warranted.

Effects of phenylethyl isothiocyanate on early molecular events in N-nitrosomethylbenzylamine-induced cytotoxicity in rat esophagus.

There is little information on early molecular events in the development of N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal tumorigenesis and of the effects of chemopreventive agents on these events. In this study, we identified genes in rat esophagus that were differentially expressed in response to short-term NMBA treatment and modulated by cotreatment with phenylethyl isothiocyanate (PEITC). Rats were fed AIN-76A diet or AIN-76A diet containing PEITC for 3 weeks. During the 3rd week of dietary treatment, they were administered three s.c. doses of NMBA (0.5 mg/kg body weight). Rats were sacrificed 24 h after the last treatment; esophagi were excised and processed for histologic grading, microarray and real-time PCR analysis. Histopathologic analysis showed that treatment of rats with PEITC had a protective effect on NMBA-induced preneoplastic lesions in the rat esophagus. We identified 2,261 genes that were differentially expressed in the NMBA-treated versus control esophagi and 1,936 genes in the PEITC + NMBA versus NMBA-treated esophagi. The intersection of these two sets resulted in the identification of 1,323 genes in NMBA-treated esophagus, the vast majority of which were modulated by PEITC to near-normal levels of expression. Measured changes in the expression levels of eight selected genes were validated using real-time PCR. Results from 12 microarrays indicated that PEITC treatment had a genome-wide modulating effect on NMBA-induced gene expression. Samples obtained from animals treated with PEITC alone or cotreated with PEITC + NMBA were more similar to controls than to samples treated with NMBA alone.

Targeting lonidamine to mitochondria mitigates lung tumorigenesis and brain metastasis.

Lung cancer often has a poor prognosis, with brain metastases a major reason for mortality. We modified lonidamine (LND), an antiglycolytic drug with limited efficacy, to mitochondria-targeted mito-lonidamine (Mito-LND) which is 100-fold more potent. Mito-LND, a tumor-selective inhibitor of oxidative phosphorylation, inhibits mitochondrial bioenergetics in lung cancer cells and mitigates lung cancer cell viability, growth, progression, and metastasis of lung cancer xenografts in mice. Mito-LND blocks lung tumor development and brain metastasis by inhibiting mitochondrial bioenergetics, stimulating the formation of reactive oxygen species, oxidizing mitochondrial peroxiredoxin, inactivating AKT/mTOR/p70S6K signaling, and inducing autophagic cell death in lung cancer cells. Mito-LND causes no toxicity in mice even when administered for eight weeks at 50 times the effective cancer inhibitory dose. Collectively, these findings show that mitochondrial targeting of LND is a promising therapeutic approach for investigating the role of autophagy in mitigating lung cancer development and brain metastasis.