Phosphomimetic Dicer S1016E triggers a switch to glutamine metabolism in gemcitabine-resistant pancreatic cancer.

OBJECTIVE: Dicer is an enzyme that processes microRNAs (miRNAs) precursors into mature miRNAs, which have been implicated in various aspects of cancer progressions, such as clinical aggressiveness, prognosis, and survival outcomes. We previously showed that high expression of Dicer is associated with gemcitabine (GEM) resistance in pancreatic ductal adenocarcinoma (PDAC); thus, in this study, we aimed to focus on how Dicer is involved in GEM resistance in PDAC, including cancer prognosis, cell proliferation, and metabolic regulation. METHODS: We generated stable shRNA knockdown of Dicer in GEM-resistant PANC-1 (PANC-1 GR) cells and explored cell viability by MTT and clonogenicity assays. Metabolomic profiling was employed to investigate metabolic changes between parental cells, PANC-1, and PANC-1 GR cells, and further implied to compare their sensitivity to the glutaminase inhibitor, CB839, and GEM treatments. To identify putative phosphorylation site involves with Dicer and its effects on GEM resistance in PDAC cells, we further generated phosphomimetic or phosphomutant Dicer at S1016 site and examined the changes in drug sensitivity, metabolic alteration, and miRNA regulation. RESULTS: We observed that high Dicer levels in pancreatic ductal adenocarcinoma cells were positively correlated with advanced pancreatic cancer and acquired resistance to GEM. Metabolomic analysis indicated that PANC-1 GR cells rapidly utilised glutamine as their major fuel and increased levels of glutaminase (GLS): glutamine synthetase (GLUL) ratio which is related to high Dicer expression. In addition, we found that phosphomimetic Dicer S1016E but not phosphomutant Dicer S1016A facilitated miRNA maturation, causing an imbalance in GLS and GLUL and resulting in an increased response to GLS inhibitors. CONCLUSION: Our results suggest that phosphorylation of Dicer on site S1016 affects miRNA biogenesis and glutamine metabolism in GEM-resistant pancreatic cancer.

Eicosapentaenoic Acid Inhibits KRAS Mutant Pancreatic Cancer Cell Growth by Suppressing Hepassocin Expression and STAT3 Phosphorylation.

BACKGROUND: The oncogenic Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation was reported to be the signature genetic event in most cases of pancreatic ductal adenocarcinoma (PDAC). Hepassocin (HPS/FGL1) is involved in regulating lipid metabolism and the progression of several cancer types; however, the underlying mechanism of HPS/FGL1 in the KRAS mutant PDAC cells undergoing eicosapentaenoic acid (EPA) treatment remains unclear. METHODS: We measured HPS/FGL1 protein expressions in a human pancreatic ductal epithelial (HPNE) normal pancreas cell line, a KRAS-wild-type PDAC cell line (BxPC-3), and KRAS-mutant PDAC cell lines (PANC-1, MIA PaCa-2, and SUIT-2) by Western blot methods. HEK293T cells were transiently transfected with corresponding KRAS-expressing plasmids to examine the level of HPS expression with KRAS activation. We knocked-down HPS/FGL1 using lentiviral vectors in SUIT-2 cells and measured the cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clonogenicity assays. Furthermore, a lipidomic analysis was performed to profile changes in lipid metabolism after HPS/FGL1 knockdown. RESULTS: We found that the HPS/FGL1 level was significantly upregulated in KRAS-mutated PDAC cells and was involved in KRAS/phosphorylated (p)-signal transduction and activator of transcription 3 (STAT3) signaling, and the knockdown of HPS/FGL1 in SUIT-2 cells decreased cell proliferation through increasing G2/M cell cycle arrest and cyclin B1 expression. In addition, the knockdown of HPS/FGL1 in SUIT-2 cells significantly increased omega-3 polyunsaturated fatty acids (PUFAs) and EPA production but not docosahexaenoic acid (DHA). Moreover, EPA treatment in SUIT-2 cells reduced the expression of de novo lipogenic protein, acetyl coenzyme A carboxylase (ACC)-1, and decreased p-STAT3 and HPS/FGL1 expressions, resulting in the suppression of cell viability. CONCLUSIONS: Results of this study indicate that HPS is highly expressed by KRAS-mutated PDAC cells, and HPS/FGL1 plays a crucial role in altering lipid metabolism and increasing cell growth in pancreatic cancer. EPA supplements could potentially inhibit or reduce ACC-1-involved lipogenesis and HPS/FGL1-mediated cell survival in KRAS-mutated pancreatic cancer cells.

NF-κB-driven suppression of FOXO3a contributes to EGFR mutation-independent gefitinib resistance.

Therapy with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs, such as gefitinib or erlotinib) significantly prolongs survival time for patients with tumors harboring an activated mutation on EGFR; however, up to 40% of lung cancer patients exhibit acquired resistance to EGFR-TKIs with an unknown mechanism. FOXO3a, a transcription factor of the forkhead family, triggers apoptosis, but the mechanistic details involved in EGFR-TKI resistance and cancer stemness remain largely unclear. Here, we observed that a high level of FOXO3a was correlated with EGFR mutation-independent EGFR-TKI sensitivity, the suppression of cancer stemness, and better progression-free survival in lung cancer patients. The suppression of FOXO3a obviously increased gefitinib resistance and enhanced the stem-like properties of lung cancer cells; consistent overexpression of FOXO3a in gefitinib-resistant lung cancer cells reduced these effects. Moreover, we identified that miR-155 targeted the 3'UTR of FOXO3a and was transcriptionally regulated by NF-κB, leading to repressed FOXO3a expression and increased gefitinib resistance, as well as enhanced cancer stemness of lung cancer in vitro and in vivo. Our findings indicate that FOXO3a is a significant factor in EGFR mutation-independent gefitinib resistance and the stemness of lung cancer, and suggest that targeting the NF-κB/miR-155/FOXO3a pathway has potential therapeutic value in lung cancer with the acquisition of resistance to EGFR-TKIs.