Adipokine Apelin/APJ Pathway Promotes Peritoneal Dissemination of Ovarian Cancer Cells by Regulating Lipid Metabolism

Abstract

Adipose tissue, which can provide adipokines and nutrients to tumors, plays a key role in promoting ovarian cancer metastatic lesions in peritoneal cavity. The adipokine apelin promotes ovarian cancer metastasis and progression through its receptor APJ, which regulates cell proliferation, energy metabolism, and angiogenesis. The objective of this study was to investigate the functional role and mechanisms of the apelin-APJ pathway in ovarian cancer metastasis, especially in context of tumor cell-adipocyte interactions. When co-cultured in the conditioned media (AdipoCM) derived from 3T3-L1 adipocytes, which express and secrete high apelin, human ovarian cancer cells with high APJ expression showed significant increases in migration and invasion in vitro. We also found that cells expressing high levels of APJ had increased cell adhesion to omentum ex vivo, and preferentially "home-in" on the omentum in vivo. These apelin-induced pro-metastatic effects were reversed by APJ antagonist F13A in a dose-dependent manner. Apelin-APJ activation increased lipid droplet accumulation in ovarian cancer cells, which was further intensified in the presence of AdipoCM and reversed by F13A or APJ knockdown. Mechanistically, this increased lipid uptake was mediated by CD36 upregulation via APJ-STAT3 activation, and the lipids were utilized in promoting fatty acid oxidation via activation of AMPK-CPT1a axis. Together, our studies demonstrate that adipocyte-derived apelin activates APJ-expressing tumor cells in a paracrine manner, promoting lipid uptake and utilization and providing energy for ovarian cancer cell survival at the metastatic sites. Hence, the apelin-APJ pathway presents a novel therapeutic target to curb ovarian cancer metastasis. IMPLICATIONS: Targeting the APJ pathway in high-grade serous ovarian carcinoma is a novel strategy to inhibit peritoneal metastasis.

Fig 1.. Apelin-derived from adipocytes promotes migration in APJ-dependent manner.

(A) Representative western blot for apelin performed on whole cell lysates obtained from pre-adipocytes and mature adipocytes. (B) Apelin secreted by pre-adipocytes and mature adipocytes into the medium quantified using ELISA. (C) Representative images of 8 hr-transwell migration assay performed in OVCAR-5-EV and APJ lines and (D) its quantification. (E) Representative images of 8 hr-transwell migration assay performed in OVCAR-8 cell and (F) its quantification. The cells were allowed to migrate to control media (10% FBS) or Adipocyte-conditioned media (Adipo CM), as indicated. Results were obtained from 3 independent experiments (mean±SEM). Statistical analysis was performed using student’s t-test for B, and one-way ANOVA followed by Tukey post hoc test for D,F. *P< 0.05; **P< 0.01;***P<0.001; ****P<0.0001.

Fig 2.. Apelin-mediated APJ activation promotes omental adhesion & invasion.

(A) Representative images of omental adhesion assay performed ex vivo in OVCAR-5-EV and APJ cell lines treated with F13A (4 ng/mL) and (B) its quantification. (C) Representative images of 16 hr-transwell invasion assay performed in OVCAR-5-EV and APJ cell and (D) its quantification. (E) Representative images of 16 hr-invasion assay performed in OVCAR-8 cell and (F) its quantification. The cells were allowed to invade towards control media (10% FBS) or Adipocyte-conditioned media (Adipo CM), as indicated. Results were obtained from 2 independent experiments in A and 3 independent experiments in C,E (mean±SEM). Statistical analysis was performed using one-way ANOVA followed by Tukey post hoc test for B,D,F. *P< 0.05; **P< 0.01;***P<0.001; ****P<0.0001.

Fig 3.. Apelin/APJ Pathway Promotes Lipid Droplet Accumulation in OvCa cells.

(A) Representative images of 24 hr lipid droplet accumulation in OVCAR-5-EV and APJ cells treated with F13A by confocal microscopy and (B) its quantification. (C) Representative images of 24 hr lipid droplet accumulation in OVCAR-8 cells treated with F13A by confocal microscopy and (D) its quantification. The cells were cultured either in serum free medium (SFM) or in adipocyte conditioned medium (Adipo CM) as indicated. F13A dose used in these experiments ranged from 10–25 ng/mL. Results were obtained from 3 independent experiments (mean±SEM). Statistical analysis was performed using one-way ANOVA followed by Tukey post hoc test for B,D. ****P<0.0001. Scale bar: 50μm.

Fig 4.. CD36 is upregulated in OvCa cells via STAT3 activation in apelin/APJ-dependent manner.

(A,B) qRT-PCR performed using cDNA obtained from (A) OVCAR-5-EV and APJ cells and (B) OVCAR-8 cells in the absence and presence of F13A treatment. (C, D) Western blots for CD36 performed on whole cell lysates (WCLs) obtained from (C) OVCAR-5-EV and APJ cells, and (D) OVCAR-8 cells. (E,F) Representative western blots and quantification for p-STAT3 and total STAT3 levels performed on WCLs obtained from (E) OVCAR-5-EV and APJ cells, and (F) OVCAR-8 cells. The cells were cultured either in serum free medium (SFM) or in adipocyte conditioned medium (Adipo CM) as indicated. F13A dose used in these experiments ranged from 10–25 ng/mL. Results were obtained from 3 independent experiments (mean±SEM). Statistical analysis was performed using one-way ANOVA followed by Tukey post hoc test for A,B. *P< 0.05; **P< 0.01; ****P<0.0001.

Fig 5.. APJ pathway promotes fatty acid utilization in OvCa cells.

(A) Fuel dependency for oxidation of glucose, glutamine, and fatty acids in OVCAR-5-EV and APJ cells as quantified by Seahorse Metabolic Analyzer. (B) Fuel flexibility for oxidation of glucose, glutamine, and fatty acids in OVCAR-5-EV and APJ cells. (C,D) Oxygen consumption rate (OCR) in OVCAR-5-EV and APJ cells when treated with (C) BSA and palmitate, and (D) BSA and palmitate in presence of CPT1a inhibitor etomoxir (40 μM) as quantified by Seahorse Metabolic Analyzer. Quantification of (E) Basal respiration rates, (F) Mitochondrial ATP production rate (oligomycin [oligo]-sensitive respiration), and (G) Maximal respiration rate (induced by FCCP, uncoupler of mitochondrial oxidative phosphorylation [OXPHOS]) in OVCAR-5-APJ cells in presence of etomoxir (40 μM). Results were obtained from 2 independent experiments (mean±SEM). Statistical analysis was performed using one-way ANOVA followed by Tukey post hoc test for A,B,E-G. *P< 0.05; **P< 0.01; ***P< 0.001; ****P<0.0001.

Fig 6.. APJ activation promotes fatty acid utilization via AMPK-CPT1a pathway.

(A,B) Western blots performed for pAMPK and total AMPK levels on whole cell lysates (WCLs) obtained from (A) OVCAR-5-EV and APJ cells, and (B) OVCAR-8 cells in the absence and presence of F13A treatment. (C,D) qRT-PCR performed for CPT1a expression using cDNA obtained from (C) OVCAR-5-EV and APJ cells and (D) OVCAR-8 cells with F13A treatment. The cells were cultured either in serum free medium (SFM) or in adipocyte conditioned medium (Adipo CM) as indicated. F13A dose used in these experiments ranged from 10–50 ng/mL. (E) Graphical summary of the paper illustrating that adipocyte-derived apelin promotes metastasis by modulating fatty acid uptake and utilization in OvCa cells. Results were obtained from 3 independent experiments (mean±SEM). Statistical analysis was performed using one-way ANOVA followed by Tukey post hoc test for C,D. *P< 0.05; **P< 0.01; ****P<0.0001.