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 pro-anthocyanidin 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.
Esophageal adenocarcinoma (EAC) is a devastating cancer characterized by rising incidence and poor prognosis. The five-year survival rate is 20% due to late stage diagnosis and ineffective treatment. It is estimated that only 15% of EAC patients have a complete histopathological response to standard chemotherapy. Thus, new strategies are urgently needed to improve therapeutic efficacy and patient survival. Our recent analysis of prechemotherapy endoscopic biopsies of EAC patients revealed differential response to neoadjuvant therapy. Differential response was reflected by two-year mortality of 9.1% in therapy responsive patients (Complete responders, no residual tumor), 22.2% in strong responders (improved TNM), 25.0% in intermediate responders (stable TNM), 42.9% in poor responders (worse TNM) and 77.8% in non-responders (worse TNM and metastasis). Transcriptomic analysis revealed differential response to therapy is linked to immune defense, inflammation (IL-4, IFNs), cell adhesion, signal transduction and angiogenesis regulation. Similar pathways were mitigated by cranberry proanthocyanidins (C-PAC) in a rat surgical model for EAC. Herein, we investigated C-PAC pretreatment alone or in combination with Paclitaxel and Carboplatin (chemo drug) on EAC cell viability (Calcein-AM), gene expression (RNA-seq) and select protein levels (Western blot). C-PAC pre-treatment and co-treatment enhance chemotherapy-induced EAC cell death, with the greatest synergistic effects in chemo-resistant OE33 cells. C-PAC pretreatment followed by chemo drugs decreased cell viability by an additional 50% and 17% compared to chemo drugs alone in OE33 cells and OE19 cells, respectively. Greater reductions were noted with pretreatment of C-PAC, followed by co-treatment with chemo drugs (64.9% in OE33 and 49.4% in OE19 cells). C-PAC enhanced chemo drug-induced EAC cell death via increased DNA damage, apoptosis and ECM degradation as evidenced by induction of phosphorylated H2AX and cleaved caspase-3, as well as inhibition of BCLXL and MMP9. C-PAC also mitigated elevated levels of P53, a gene with a well-documented role in EAC progression and therapeutic resistance. Transcriptome analysis of cells treated with C-PAC and chemo drugs is underway and will be compared to signaling networks differentially expressed in patients stratified by treatment responsiveness. Results suggest that C-PAC pretreatment may offer a non-toxic intervention strategy for enhancing chemotherapeutic efficacy. Citation Format: Yun Zhang, Katherine Weh, Connor Howard, Kiran Lagisetty, Dyke McEwen, Jules Lin, Rishindra M. Reddy, Andrew Chang, David G. Beer, Amy Howell, Laura A. Kresty. Proanthocyanidins enhance chemotherapy-induced esophageal adenocarcinoma cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1418.
Esophageal adenocarcinoma (EAC), the major subtype of esophageal cancer in the US, is characterized by increasing incidence rates and high mortality due to ineffective screening, late-stage diagnosis, and poor treatment efficacy. The only known precursor to EAC is Barrett’s Esophagus (BE), but the precise mechanisms and molecular events leading to disease progression are still being unraveled. In recent years, analysis of TCGA data revealed that isoform switching events with predicted functional consequences are common in many cancers, but the characterization of switching events in esophageal adenocarcinoma (EAC) is lacking. Next-generation sequencing was utilized to detect levels of RNA transcripts and specific isoforms in 66 treatment naïve esophageal tissues from 41 patients. Tissues ranged from premalignant Barrett’s esophagus (BE), BE with low or high-grade dysplasia (LGD or HGD) to EAC. Samples were stratified by histopathology, p53 mutation status, and significant isoform switching events with predicted functional consequences, followed by enrichment analysis. Barrett’s with high-grade dysplasia increased risk for EAC progression. Comparing BE with LGD and BE with HGD, isoform switching events were identified in 75 genes including KRAS, APC, RNF-128 and WRAP53. Stratification based on p53 status increased the number of significant isoform switches to 135 suggesting switching events impact cellular functions based on tissue histopathology, as well as p53 status. Analysis of isoforms agnostic to mutant p53, exclusive to mutant p53, and shared between the two groups revealed unique signatures including demethylation, lipid and retinoic acid metabolism, and glucuronidation, respectively. About 25% of isoform switching events were identified without significant alteration of gene-level expression. Importantly, two p53-interacting isoforms, RNF128 and WRAP53, were significantly linked to patient survival. Analysis of global isoform switching consequences and alternative splicing events also revealed significant changes in the fraction of coding transcripts, complete open-reading-frames, signal peptides, alternative 5’ donor sites, and alternative transcription start sites. Thus, analysis of isoform switching events may provide new insight for the identification of prognostic markers and inform new potential therapeutic targets for EAC treatment or prevention. Citation Format: Yun Zhang, Katherine M. Weh, Connor L. Howard, Jean-Jack Riethoven, Jennifer L. Clarke, Kiran H. Lagisetty, Jules Lin, Rishindra M. Reddy, Andrew C. Chang, David G. Beer, Laura A. Kresty. Characterizing isoform switching events in esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5741.
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