(T1030-05-27) Tamoxifen and Metabolite Effects on Cellular Metabolism in Pancreatic Cancer Cell Lines

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy and the 3rd leading cause of cancer-related mortality in the United States, with a 5-year survival rate of only 13%.¹ Despite groundbreaking progress in cancer management, effective treatment of PDAC is still lacking. Most modern anticancer approaches fail due to the chemotherapy-resistant, refractory, and highly aggressive nature of the PDAC. Studies have reported the presence of estrogen receptors (ER)- ERα and ERβ, and their role in tumorigenesis of several types of pancreatic neoplasms.² In this context, several clinical studies in pancreatic cancer patients were conducted using tamoxifen, a selective estrogen receptor modulator, which resulted in moderate increase in median overall survival.³ However, there is debate whether this efficacy is due to antiestrogenic effects of tamoxifen or its non-estrogenic off-target effects. Tamoxifen has also demonstrated anti-tumor effects in ER-negative tumors via non-ER mediated mechanisms.⁴ These mechanisms include the inhibition of protein kinase C, a key regulator of intracellular signal-transducing networks, and also a general antiproliferative and pro-apoptotic effect that helps reduce the neovascularization, metastasis, and multidrug resistance of tumor cells.³ Furthermore, TAM and romidepsin, an inhibitor of histone deacetylase, synergistically induced senescence in pancreatic cancer cells by promoting reactive oxygen species and mitochondrial lipid peroxidation while downregulating FOXM1 expression, which together significantly inhibited tumor growth in CFPAC-1 xenograft models.⁵ Therefore, it is important to determine the changes in cellular metabolism caused by tamoxifen in order to understand its exact molecular mechanism in pancreatic cancer cell lines. Additionally, tamoxifen also undergoes extensive primary and secondary metabolism by CYP450 enzymes in the liver to generate its active metabolites 4-hydroxytamoxifen and endoxifen.⁶ These metabolites have up to 100-fold greater affinity for the ER and 30 to 100-fold greater potency in suppressing estrogen-dependent cell proliferation compared to tamoxifen.⁷. Thus, this study aims to (i) elucidate the role of tamoxifen and its active metabolites in cellular metabolism of pancreatic cancer cell lines using LC-MS/MS based metabolomics and (ii) compare the metabolic changes, differentially expressed metabolites, and altered metabolic pathways associated with tamoxifen, 4-hydroxytamoxifen and endoxifen treatment.

Methods: Two human pancreatic cell lines, PANC-1 and CFPAC-1 were utilized for all experiments. PANC-1 cells were plated, and 18 hours later, cells were treated with 5 µM tamoxifen, 1 µM endoxifen, and 1 µM 4-hydroxytamoxifen separately. In the case of CFPAC-1, the cells were treated with 5 µM tamoxifen, 0.05 and 1.0 µM endoxifen, and 0.05 and 1.0 µM 4-hydroxytamoxifen. Cells were incubated for 48 hours and harvested using 80% methanol for metabolite isolation. The scraped cell samples were centrifuged to precipitate the cells, supernatants were collected and dried using nitrogen gas. The dried extracts were then reconstituted, and the samples were analyzed in Shimadzu 8060 NX using previously developed and validated LC-MS/MS assays that account for 120 metabolites. The method validation was done following the FDA’s M10 bioanalytical method validation and study sample analysis guidelines.⁸ All analysis were done in four biological replicates. Lab Solutions software was used to integrate metabolite concentrations and peak areas, which were then normalized to their respective cell counts. Metaboanalyst 6.0 (https://www.metaboanalyst.ca/) was used to analyze the normalized values. Differences between the control and treatment groups were compared using Student’s t-test where p < 0.05 was considered significant.

Results: Treatment with tamoxifen, endoxifen, and 4-hydroxytamoxifen in the CFPAC-1 cell line led to distinct metabolic alterations that are shown in the partial least squares discriminant analysis (PLS-DA) plot (Figure 1). Upon treatment with tamoxifen, endoxifen, and 4-hydroxytamoxifen, 17, 25, and 52 metabolites respectively, showed statistically significant changes, among which the top differentially altered metabolites are depicted in the heatmap (Figure 2). Following drug treatment, we observed the downregulation of key metabolites involved in tumorigenesis as depicted in the heatmap. Several key amino acids that are vital in tumorigenesis and tumor immunity by acting as nutrients, signaling molecules, and regulators of epigenetic modification and gene transcription were also downregulated.⁹ The differentially altered metabolites were used to perform pathway analysis, which predicted the citrate cycle, pyruvate metabolism, and biosynthesis pathway of key amino acids such as arginine, alanine, aspartate, glutamate, etc. to be most downregulated (Figure 3). In the case of the PANC-1 cell line, drugs did not show any significant metabolic alteration.

Conclusion: Treatment with tamoxifen and its active metabolites led to distinct alterations of the metabolome in the pancreatic cancer cells. We found surprising contrasts among the significantly altered metabolites caused by the drugs in CFPAC-1 and PANC1 cells. A prior study reported a significantly higher ERβ/ERα ratio in CFPAC-1 (5.49) cells compared to PANC-1 cells (0.38).¹⁰ We hypothesize that the increased metabolic alterations observed in CFPAC-1 cell lines might be due to its higher ERβ/ERα ratio which has impact on estrogen-mediated growth stimulation. This is the first study to use LC-MS/MS based metabolomics for elucidating cellular metabolite alterations by tamoxifen and its metabolites in pancreatic cancer cells.

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Figure 1. Partial Least Squares Discriminant Analysis (PLS-DA) plot showing the distinct metabolic alterations upon treatment of CFPAC-1 cells with 5 µM tamoxifen, 1 µM endoxifen and 1 µM 4-hydroxytamoxifen. Green circles indicate drug-treated conditions and red circles indicate DMSO-treated controls (n=4).


Figure 2. Heatmap representing the differentially altered metabolites upon treatment of CFPAC-1 cells with 5 µM tamoxifen, 1 µM endoxifen, and 1 µM 4-hydroxytamoxifen. The green class indicates drug-treated condition and the red class indicates DMSO-treated controls (n=4).


Figure 3. Pathway enrichment analysis for human KEGG pathways significantly altered upon treatment of CFPAC-1 cells with 5 µM tamoxifen, 1 µM endoxifen, and 1 µM 4-hydroxytamoxifen.