MOLECULAR MECHANISM OF THE CYTOTOXIC AND IMMUNOSTIMULATORY ACTIVITY OF 15 DEOXY PROSTAMIDE J2: INVESTIGATION OF CHOP10 AND NF?B SIGNALING IN COLON CANCER

ECU Author/Contributor (non-ECU co-authors, if there are any, appear on document)

Hussam Albassam (Creator)

Institution

East Carolina University (ECU )

Web Site: http://www.ecu.edu/lib/

Abstract: The endoplasmic reticulum (ER) stress pathway is a prominent regulator of cancer cell death and as a result, ER stress inducers are being exploited pharmacologically. Cytotoxic ER stress is typically regulated by the transcription factor, C/EBP homologous protein 10 (CHOP10). The products of CHOP10 transcription include three pro-apoptotic proteins\; tribbles-related protein 3 (TRB3), death receptor-5 (DR5), and ER oxidoreductase 1[alpha] (ERO1[alpha]). Our previous findings showed that cell death induced by 15deoxy, [delta]12,14 prostamide J2 (15d PMJ2) occurred in an ER stress-dependent manner. However, the signaling pathway that regulates15d PMJ2-mediated ER stress cell death has not been identified. Therefore, the goal of this study is to determine the role of CHOP10 and its transcriptional products in cytotoxic ER stress that is induced by 15d PMJ2 in colon cancer cells. Our results show that 15d PMJ2 exhibited a preferential cytotoxicity toward the human colon cancer cell line, HCT116, compared to the non-tumorigenic colon cell line, FHC. The induction of cell death in HCT116 cells was accompanied by a significant increase in CHOP10 protein expression. In addition, pharmacological blockade of ER stress prevented 15d PMJ2 induced cell death indicating that cell death is reliant upon ER stress. To determine the role of CHOP10 in this process, a genetic deletion of CHOP10 (CHOP10-KO) was performed using CRISPR/Cas9. 15d PMJ2 induced death in WT but not in CHOP10-KO cells. Interestingly, TRB3 synthesis appears to require 15d PMJ2-induced CHOP10 expression. Therefore, the activity of protein kinase B (PKB)/Akt, a known TRB3 target, was investigated. CHOP10 stimulation by 15d PMJ2 inactivated Akt in WT but not in CHOP10-KO cells. TRB3 transfected CHOP10-KO cells restored 15d PMJ2-induced death, suggesting the importance of TRB3 in CHOP10-initiated inhibition of Akt and the control of the ER stress-mediated cell death by 15d PMJ2. Moreover, PD-L1, which is a T cell inhibitory ligand, is downstream of Akt, thus cell surface PD-L1 expression was examined. 15d PMJ2 decreased PD-L1 expression in WT cells and it was comparable to CHOP10-KO cells. The activity of NF-kB, which is a modulator of PD-L1 expression, was investigated. A decrease in PD-L1 expression was mediated by an inhibition of NF-kB activity by 15d PMJ2, and NF-kB transfection mitigated 15d PMJ2-reduced PD-L1 expression. In 15d PMJ2 treated cells, a decrease in PD-L1 expression reduced apoptosis of T cells, indicating that 15d PMJ2 enhanced anti-tumor immunity via PD-L1 down-regulation. Thus, 15d PMJ2 is a potential anti-tumor agent that possesses the dual actions of tumor destruction and anti-tumor immune cell stimulation.

Additional Information

Publication

Dissertation

Language: English

Date: 2023

Subjects

ER stress;cell death;prostaglandins;CHOP10;NF-kB;immunostimulantory;PD-L1

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