Abstract 4811: Functional pathway analysis in pancreatic cancer cells treated with a novel anti-cancer agent, copper-tolfenamic acid

Anti-cancer activity of tolfenamic acid (TA) has been studied using several preclinical cancer models including pancreatic cancer (PaCa). Since the dosage used for the anti-cancer actions of TA is rather high, we investigated Copper-TA (Cu-TA) for anti-proliferative activity using 12 cancer cells (representing 6 cancers) and demonstrated that Cu-TA is more effective than TA (IC50 values are 30-80% less than TA). Recently, we reported that Cu-TA inhibits tumor growth in mouse xenograft (pancreatic tumor) model. The objective of this investigation is to elucidate the underlying mechanisms of Cu-TA using PaCa cell lines. MIA PaCa-2 cells were treated with vehicle (dimethyl sulfoxide) or 29 µM (IC50) of Cu-TA and processed by next-generation sequencing (NGS) to determine the differentially expressed genes using Oncology Biomarker (HTG EdgeSeq) panel. The functional significance of the altered gene expression was determined via Ingenuity Pathway Analysis which identified several networks, regulators, as well as molecular and cellular functions that were affected by Cu-TA treatment. A total of 18 networks were found to be involved with Cu-TA treatment and these networks had a significant overlap. Confirmation experiments assessing the alterations in the expression of selected candidate markers were conducted by qPCR and Western blot analysis using both MIA PaCa-2 and PANC 1 cell lines. The top upstream regulators included tumor protein p53, human epidermal receptor growth factor 2, Sp1 and signal transducer and activator of transcription 3. These regulators were confirmed by Western blot analysis. The top five molecular/cellular functions affected by Cu-TA treatment were cell death/survival, cellular development, cell growth/proliferation, cell cycle and cellular movement. qPCR results of selected genes, Centromere protein F, DNA damage inducible transcript 3 and S-phase kinase-associated protein 2 (differentially expressed genes in sequencing) demonstrated that Cu-TA is efficacious at lower doses than TA. In summary, the NGS and Ingenuity Pathway Analysis identified critical genes or pathways altered by Cu-TA. This investigation demonstrated that the networks and regulators associated with cancer cell survival or apoptosis that are modulated in PaCa cells suggests that Cu-TA is altering genes associated with cancer, thereby demonstrating its potential as an effective anti-cancer agent. Citation Format: Myrna Hurtado, Laszlo Prokai, Umesh T. Sankpal, Blair Leves