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

Abstract

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.

Keywords: KRAS mutation; eicosapentaenoic acid; hepassocin; pancreatic cancer.

Figure 1

Expression level of hepassocin (HPS) in Kirsten rat sarcoma viral oncogene homolog (KRAS)-mutant pancreatic cancer cells. (A) Protein expressions of HPS and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by hTERT-HPNE, BxPC-3, PANC-1, MIA Paca-2, and SUIT-2 cells were measured by a Western blot analysis. GAPDH was used as the internal control. (B) Protein expressions of phosphorylated (p)-STAT3, STAT3, and HPS by HEK293T cells transiently transfected with the RAS wild-type (WT), constitutively active RAS (V12), and dominant negative RAS (N17) plasmids and a control vector were measured by a Western blot analysis. Actin was used as the internal control. The p-STAT3/STAT3 ratio (C) and HPS (D) expression level of indicated cells were analyzed with ImageJ software, normalized to actin, and evaluated as multiples of change compared to vector-transfected cells. Results are shown as the mean ± SD of three independent experiments. ** p < 0.01 by a one-way ANOVA followed by Tukey’s post-hoc test.

Figure 2

Knockdown of hepassocin (HPS) decreases pancreatic cancer cell growth in SUIT-2 cells. (A) SUIT-2 cells were infected with a lentivirus carrying the short hairpin (sh) control vectors of Scramble, Void, and pLKO, and shHPS vectors #20, #21, and #90, and protein expressions of STAT3, phosphorylated (p)-STAT3, HPS, and GAPDH of stably transfected SUIT-2 cells were measured by a Western blot analysis. GAPDH was used as the internal control. (B) HPS expression levels of the indicated SUIT-2 cells were analyzed with ImageJ software, normalized to GAPDH, and evaluated as multiples of change compared to SUIT-2 cells. Results are shown as the mean ± SD of three independent experiments. * p < 0.05 vs. Scramble, ^# p < 0.05 vs. Void, and ^† p < 0.05 vs. pLKO control (by a one-way ANOVA followed by Tukey’s post-hoc test). (C) The indicated SUIT-2 cells were seeded and cultured for a period of time (24, 48, 72, and 96 h), and cell viability was subsequently evaluated by an MTT assay. Multiple changes of cell viability were compared to each 24-h group. Results are shown as the mean ± SD of three independent experiment, each performed in triplicate. * p < 0.05 vs. Scramble, ^# p < 0.05 vs. Void, and ^† p < 0.05 vs. pLKO control (by a one-way ANOVA followed by Tukey’s post-hoc test). (D) The cell clonogenicity effect of HPS knockdown on SUIT-2 cells was measured by a clonogenic assay. (E) Total cell numbers were measured as the number of colonies Size (pixel*2) = 100-infinity calculated by ImageJ. * p < 0.05 vs. the Scramble control.

Figure 3

Knockdown of hepassocin (HPS) induces cell cycle arrest in SUIT-2 cells. (A) SUIT-2 cells were infected with a lentivirus carrying control vectors of Scramble and Void and the short hairpin (sh) HPS vectors #21 and #90, the indicated cell pellets were fixed with 60% EtOH at 4 °C and stained with propidium iodide and RNase A, and their DNA contents were analyzed by flow cytometry. (B) G₀/G₁, S, and G₂/M phase percentages of the indicated SUIT-2 cells were determined by FlowJo software using the Dean–Jett–Fox model (w/sync. peak). (C) Cyclin D, B, and E expression levels of the indicated SUIT-2 cells were analyzed by Western blotting and quantified with ImageJ software, normalized to tubulin, and evaluated as multiples of change compared to SUIT-2 cells. Results are shown as the mean ± SD of three independent experiments. * p < 0.05 vs. SUIT-2 (by a one-way ANOVA followed by Tukey’s post-hoc test).

Figure 4

Knockdown of hepassocin (HPS) in SUIT-2 cells changes the lipid profile. (A) Expression levels of α-linolenic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid (LA), and arachidonic acid (AA). (B) Abundances of hydroxyeicosapentaenoic acid (HEPE), lipoxin, hydroperoxyeicosapentaenoic acid (HpEPE), Resolvin E1, and Resolvin E2 in SUIT-2 cells with knockdown of HPS. Control vectors of Scramble and Void, and short hairpin (sh) HPS vectors #21 and #90. Results are shown as the mean ± SD of three independent experiments. * p < 0.05 vs. the control (by a one-way ANOVA followed by Tukey’s post-hoc test).

Figure 5

Treatment with n-3 and n-6 polyunsaturated fatty acids (PUFAs) decreases the viability of SUIT-2 cells. SUIT-2 and hTERT-HPNE cells were exposed to different concentrations (0, 3.125, 6.25, 12.5, 25, 50, 100, and 200 μM) of n-3 or n-6 PUFAs for 48 (A) and 72 h (B), and cell viability was subsequently evaluated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Ethanol (75%) was used to prepare the solution and as serial dilution controls. The percentage of cell viability is shown relative to the untreated controls. Results are shown as the mean ± SD of three independent experiments, each performed in triplicate. * p < 0.05 vs. untreated SUIT-2 cells, ^# p < 0.05 vs. untreated hTERT-HPNE cells (by a two-way ANOVA followed by Tukey’s post-hoc test). (C) Protein expressions of signal transduction and activator of transcription 3 (STAT3), phosphorylated (p)-STAT3, and HPS of the indicated SUIT-2 cells were measured by a Western blot analysis. Tubulin was used as the internal control. (D) Expression levels of p-STAT3/STAT3 and HPS were analyzed with ImageJ software, normalized to actin, and evaluated as multiples of change compared to untreated cells. Results are shown as the mean ± SD of three independent experiments. * p < 0.05 (by a one-way ANOVA followed by Tukey’s post-hoc test). (E) Protein expressions of ACC-1 and FAS of the indicated SUIT-2 cells were measured by a Western blot analysis. Actin was used as the internal control. (F) Expression levels of ACC-1 and FAS were analyzed with ImageJ software, normalized to actin, and evaluated as multiples of change compared to untreated cells. Results are shown as the mean ± SD of three independent experiments. * p < 0.05 (by a one-way ANOVA followed by Tukey’s post-hoc test).