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. Author manuscript; available in PMC: 2020 Sep 15.
Published in final edited form as: Mol Cell Biochem. 2019 Jul 10;461(1-2):37–46. doi: 10.1007/s11010-019-03586-9

Adiponectin Receptor Agonist AdipoRon Induces Apoptotic Cell Death and Suppresses Proliferation in Human Ovarian Cancer Cells

Amin A Ramzan 1,*, Benjamin G Bitler 2, Douglas Hicks 2, Kelsey Barner 2, Lubna Qamar 2, Kian Behbakht 1, Theresa Powell 2, Thomas Jansson 2,*, Heidi Wilson 2
PMCID: PMC7490954  NIHMSID: NIHMS1623888  PMID: 31292831

Abstract

Objectives

We tested the hypothesis that stimulation of adiponectin receptors with the synthetic agonist AdipoRon suppresses proliferation and induces apoptotic death in human high grade serous ovarian tumor cell lines and in ex vivo primary tumors, mediated by activation of 5’ AMP-activated protein kinase (AMPK) and inhibition of mechanistic target of rapamycin (mTOR).

Methods

We determined the effect of AdipoRon on high grade serous ovarian tumor cells lines (OVCAR3, OVCAR4, A2780) and ex vivo primary tumor tissue. Western blotting analysis was performed to examine changes in activation of AMPK and mTOR signaling and flow cytometry was utilized to examine changes in cell cycle progression. Immunofluorescence of cleaved caspase-3 positive cells and flow cytometry of annexin V positive cells were used to determine changes in apoptotic response. The CyQUANT proliferation assay was used to assess cell proliferation.

Results

AdipoRon treatment increased AMPK phosphorylation (OVCAR3 P=0.01; A2780 P=0.02) but did not significantly alter mTOR activity. AdipoRon induced G1 cell cycle arrest in OVCAR3 (+12.1%, P=0.03) and A2780 (+12.0%, P=0.002) cells. OVCAR3 and OVCAR4 cells treated with AdipoRon underwent apoptosis based on cleaved caspase-3 and annexin V staining. AdipoRon treatment resulted in a dose dependent decrease in cell number versus vehicle treatment in OVCAR3 (−61.2%, P<0.001), OVCAR4 (−79%, P<0.001) and A2780 (−56.9%, P<0.001). Ex vivo culture of primary tumors treated with AdipoRon resulted in an increase in apoptosis measured with cleaved caspase-3 immunohistochemistry.

Conclusions

AdipoRon induces activation of AMPK and exhibits an anti-tumor effect in ovarian cancer cell lines and primary tumor via a mTOR-independent pathway.

Keywords: Ovarian Cancer, Adiponectin, mTORC1, AMPK

INTRODUCTION

Ovarian cancer is the most lethal gynecologic malignancy, resulting in about 14,000 deaths yearly in the United States [1]. The majority of ovarian cancer cases are diagnosed at an advanced stage, and require a combination of platinum-based systemic chemotherapy and surgical cytoreduction for initial management [2]. Unfortunately, most women with ovarian cancer experience recurrence of their disease and will subsequently receive another round of platinum-based chemotherapy. The five-year overall survival, including early stage cases, is 47%. While new therapies, including poly (ADP-ribose) polymerase (PARP) inhibitors, have emerged over the last decade, there is a clear need to identify novel targets and agents for the treatment of ovarian cancer [3].

Adiponectin is a protein hormone secreted by adipose tissue that regulates metabolism, including glucose homeostasis and fatty acid oxidation. There is a well-established inverse relationship between body mass index and circulating adiponectin levels as reflected by the typically lower adiponectin levels observed in obese individuals [4]. Adiponectin promotes insulin sensitivity in muscle and liver and low levels of adiponectin therefore contribute to the development of insulin resistance and metabolic disease [5]. In cancers that have a clear association with obesity (breast, colon and endometrial cancers), treatment of cultured tumor cells with exogenous adiponectin results in significantly reduced proliferation and increased cell death [6–8]. Obesity has been established as one of the few modifiable risk factors for ovarian cancer and among women with ovarian cancer, obese individuals have higher cancer related mortality than their normal-weight counterparts [9, 10]. Although not directly related to obesity, recent evidence suggests that patients with ovarian cancer have lower circulating levels of adiponectin compared to healthy controls with similar body mass index (BMI) [11, 12]. Collectively, these observations are consistent with the possibility that adiponectin inhibits ovarian tumor growth.

A novel small-molecule AdipoR1 and AdipoR2 agonist, AdipoRon, has recently been developed. Oral administration of AdipoRon in a mouse model of obesity-induced diabetes mellitus restored insulin sensitivity and improved survival [13]. Analysis of muscle and liver tissue from these mice revealed that AdipoRon was acting similarly to adiponectin by activating AMPK.

The intracellular effects of adiponectin are predominantly mediated through its interaction and activation of two receptors, AdipoR1 and AdipoR2, which are both expressed by ovarian cancer cells [14, 15]. We tested the hypothesis that stimulation of adiponectin receptors (AdipoR1 and AdipoR2) with the synthetic agonist AdipoRon suppresses proliferation and induces apoptotic cell death in human high grade serous ovarian tumor cell lines and in ex vivo primary tumors, mediated by activation AMPK and inhibition of mTOR signaling.

MATERIALS AND METHODS

Cell Lines and Cell Culture

Human high grade epithelial ovarian cancer cell lines OVCAR3, OVCAR4 and A2780 were obtained from Gynecologic Tumor and Fluid Bank at the University of Colorado (COMIRB #07–935). All cell lines were authenticated at the beginning of this study by short tandem repeat profiling, as described previously [16]. All three cell lines were cultured in RPMI 1640 medium (Thermo Fisher, Waltham, MA) containing 10% fetal bovine serum and supplemented with 1% penicillin and streptomycin. The cells were maintained in a humidified incubator at 37° C with 5% CO2. Cell lines were routinely tested for mycoplasma contamination.

AdipoRon treatment

Cells were plated in 96-well plates and treated with various concentrations (0–50μM) AdipoRon (Sigma-Aldrich, St. Louis, MO) over a time course of 24–48 hours.

Western Blot Analysis

Cells were harvested and lysed on ice for 15 minutes in lysis buffer (30 mM Tris-HCl pH 7.4, 150mM NaCl, 1% Triton X-100, 10% glycerol and 2mM EDTA) containing PMSF and a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). Cells were then mechanically scraped off the plate and centrifuged at 13,000 RPM for 10 minutes. Protein concentration from the supernatant was determined by the BCA protein assay reagent kit (Thermo Fisher, Waltham, MA). Cell lysates were electrophoresed on SDS-polyacrylamide gels and transferred onto PVDF membranes. Membranes were blocked with 5% bovine serum albumin in TBS-T buffer, with the exception of 4EBP1 where membranes were blocked in 5% milk/TBST Primary antibody incubations were performed overnight at 4° C using antibodies targeting p-AMPK (Thr 172, 1:1000), total p70 S6 kinase (1:1000), p-p70 S6 kinase (Thr 389, 1:1000), total p-4EBP1 (1:1000), p-4EBP1 (Thr 37/46, 1:1000), Raptor (1:1000), p-Raptor (Ser 792, 1:1000), TSC2 (1:1000), p-TSC2 (Ser 1387, 1:1000), RpS6 (1:1000), p-RpS6 (Ser 235/236, 1:1000 (Cell Signaling Technology, Beverly, MA), Anti-phospho-Ser/Thr-Pro MPM-2 (1:1000, Millipore, Burlington, MA) and β-actin (1:1000, Sigma-Aldrich, St. Louis, MO). The results were visualized with horseradish peroxidase-conjugated secondary antibodies (Sigma Aldrich, St. Louis, MO) and enhanced chemiluminescence utilizing the G:BOX system (Syngene, Frederick, MD).

Cell Cycle Analysis

One hundred thousand cells were plated in a 100 × 20mm cell culture dish. Cells were allowed to attach for 24 hours then serum starvation was performed for 6 hours to synchronize cells. Cells were then subjected to treatment with vehicle (DMSO) or AdipoRon at a dose of 50 μM. After 24 hours of treatment cells were harvested and incubated in Krishan stain (0.224 g sodium citrate 2H2O, 9.22 mg propidium iodide, 2.0 mL 1% NP40 in H2O, 2.0 mL 1 mg/mL RNase in 200 mL H20) [17]. Cells were analyzed using flow cytometry (Beckman Coulter, Brea, CA) by the University of Colorado Flow Cytometry Core Facility. Fifty thousand events were collected per sample and cell cycle analysis was performed using ModFit LT software (Verity Software House, Topsham, ME). For detection of mitotic progression by MPM-2, OVCAR3 and OVCAR4 cells were incubated with 2 mM Thymidine (Sigma Aldrich, St. Louis, MO) overnight, washed out with PBS, and subsequently incubated overnight in 2 mM Thymidine. Cells were treated with vehicle or 50 μM AdipoRon for 24 hours and protein was collected and analyzed as above.

Detection of Apoptosis by Flow Cytometry

Cells were plated in a 100 × 20mm cell culture dish. Cells were allowed to attach for 24 hours then serum starvation was performed for 6 hours to synchronize cells. Cells were then subjected to treatment with vehicle (DMSO) or AdipoRon at a dose of 50 μM. After 24 hours of treatment cells were stained with Alexa Fluor 488 annexin V (Invitrogen, Carlsbad, CA) and propidium iodide according to the manufacturer’s protocol and analyzed by flow cytometry (Beckman Coulter, Brea, CA).

Detection of Apoptosis by Capase-3 Activation Assay

Apoptosis was measured by caspase-3 activation. After treatment of tumor cells with vehicle (DMSO) or AdipoRon at a dose of 50μM for 24 hours, cells were fixed with 10 % phosphate buffered formalin (Fisher Scientific, Pittsburgh, PA, USA) at room temperature (RT) for 15 min. Cells were washed with phosphate buffered saline (PBS) and subsequently permeabilized with 0.5 % Triton X-100 for 5 min, washed with PBS, blocked with 2% bovine serum albumin (BSA) for 1 h, and treated with primary antibody directed to cleaved caspase-3 (1:400; rabbit anti-cleaved caspase-3, Cell Signaling, Danvers, MA, USA) overnight at 4 °C. Cells were washed with PBS five times before application of secondary antibody conjugated to a fluorescent probe (1:100; donkey anti-rabbit CY5, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and 5 μg/ml 4′,6-diamidina-2-phenylindole (DAPI; Sigma-Aldrich) for 45 min at room temperature followed by five washes with PBS. OPDA (20 mg/ml, o-phenylenediamine dihydrochloride in 1 M Tris, pH 8.5) was applied to slides for preservation of fluorescence and coverslip mounted. Fluorescence was imaged with a 3I MARIANAS inverted spinning disk confocal microscope (University of Colorado AMC Light Microscopy Core) and images were analyzed for percent of cell population positive for active caspase 3, using SlideBook software (Intelligent Imaging Innovations, Inc., Denver, CO, USA).

Cell Proliferation Assay

Viable cell number was assessed in triplicate with the CyQUANT cell proliferation assay kit (Thermo Fisher, Waltham, MA) in accordance with the manufacturer’s instructions. The assay utilizes a fluorescent dye to measure double strand DNA content with a microplate reader with excitation at 485 nm and emission detection at 530 nm on a SpectraMax M2 microplate reader (Molecular Devices, San Jose, CA, USA).

Gynecologic Tissue and Fluid Bank (GTFB)

The University of Colorado has an Institutional Review Board approved protocol (COMIRB #07–935) in place to collect tissue from gynecologic patients with both malignant and benign disease processes. All participants are counseled regarding the potential uses of their tissue and sign a consent form approved by the Colorado Multiple Institutional Review Board.

Ex vivo culture

Primary ovarian cancer tumor samples were obtained from the GTFB at the University of Colorado. Tumors were sectioned at 300-micron thickness using a Krumdieck Tissue Slicer (TSE Systems, Chesterfield, MO, USA). Tumor sections were cultured with vehicle control or AdipoRon (50 μM) for 24 hours. Following a 24-hour incubation, tissue sections were fixed in 10% buffered formalin, paraffin embedded and sectioned.

Immunohistochemistry

Tissue from ex vivo culture was utilized for immunohistochemistry. Four-micron thick paraffin sections were deparaffinized, antigens unmasked and stained for Cleaved Caspase-3 (Cell Signaling; Danvers, MA; rabbit polyclonal; cat#9661; 1:1000). Antigens were revealed in BORG Decloaker (Biocare Medical, Concord, CA) for 10 minutes at 110°C (NxGen Decloaker, Biocare) with an ambient cool down for 10 minutes. Immunodetection was performed on the Benchmark XT immunostainer (Ventana Medical Systems (Roche), Tucson, AZ) at an operating temperature of 37°C and primary antibodies were incubated for 32 minutes. Primary antibodies were detected with a modified I-VIEW DAB (Ventana) detection kit. The I-VIEW secondary antibody was replaced with full strength Rabbit ImmPress polymer (Vector Laboratories, Carpinteria, CA; cat# MP-7401) and the enzyme was replaced with diluted Rabbit ImmPress polymer (Vector; 50% v/v in PBS pH 7.6). All sections were counterstained in Harris hematoxylin for 2 minutes, blued in 1% ammonium hydroxide (v/v), dehydrated in graded alcohols, cleared in xylene and cover-glass mounted using synthetic resin. Staining was performed by the University of Colorado Cancer Center Histopathology Core. The slides were de-identified and a Histology score (H-score) was calculated [18]. The H-score is calculated by the intensity (0,1,2,3) and percentage of tissue with that intensity.

RESULTS

AdipoRon activates AMPK

To determine the effect of AdipoRon on cellular signaling, we first assessed AMPK phosphorylation. Treatment with 50 μM AdipoRon significantly increased phosphorylation of AMPK in OVCAR3 (P=0.01) and A2780 (P=0.02) cells when compared to treatment with vehicle (Figure 1). In OVCAR4 cells, while not statistically significant, we observed a trend that AdipoRon treatment increased AMPK phosphorylation versus vehicle treatment (Figure 1, P=0.06). Phosphorylation of AMPK is known to inhibit the pro-proliferative mTORC1 pathway in tumor cells [19]. To determine the effects of AdipoRon on mTORC1 signaling, we assessed multiple downstream functional readouts of mTORC1 activity, including p-P70 S6 kinase, p-4EBP1 and p-Ribosomal protein 6 [20]. In contrast to our hypothesis we, did not observe an inhibition of mTORC1 signaling in OVCAR3 and OVCAR4 cells treated with 50 μM AdipoRon (Figure 2A–F, I, L). In fact, in the OVCAR4 cell line, we observed a statistically significant, albeit small, increase in phosphorylation of 4EBP1 (Figure 2F). Furthermore, AdipoRon did not increase Ser-792 phosphorylation of raptor (Figure 2G,J), a component of the mTORC1 complex, or phosphorylation of Ser-1387 on TSC2 (Figure 2H,K), upstream regulator of mTORC1, both established targets of AMPK and when phosphorylated resulting in mTORC1 inhibition. Collectively these findings demonstrate that AdipoRon does not inhibit mTORC1 signaling in ovarian cancer cells.

Figure 1: Effect of AdipoRon on AMPK activity.

Figure 1:

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Protein lysates from ovarian cancer cells were generated from cells treated with vehicle control or 50 μM AdipoRon for 4 hours. Representative Western blots of phosphorylated AMPK are shown for OVCAR3, OVCAR4, and A2780 cells. β-actin was used as a loading control. Densitometry was performed to quantify changes in protein levels. Mean ± s.e.m., n=3, *p<0.005, **p<0.001 vs. vehicle treatment.

Figure 2: Effect of AdipoRon on mTORC1 signaling.

Figure 2:

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Protein lysates from ovarian cancer cells were generated from cells treated with vehicle control or 50 μM AdipoRon for 4 hours. Representative Western blots of total and phosphorylated P70 S6 kinase, 4EBP1, Raptor, TSC2 and Ribosomal Protein S6 are shown for OVCAR3 and OVCAR4 cells. Amido Black was used as a loading control. Densitometry was performed to quantify changes in protein levels. Mean ± s.e.m., n=3, *p<0.005, **p<0.001 vs. vehicle treatment.

AdipoRon induces G1 cell cycle arrest and decreases mitosis in ovarian cancer cells

We next determined the effect of AdipoRon treatment on cell cycle progression. Using flow cytometry, we examined propidium iodide incorporation and analyzed cell cycle progression in OVCAR3, OVCAR4 and A2780 cells. Compared to vehicle, OVCAR3 and A2780 cell lines demonstrated a significant accumulation of cells in the G1 phase when treated with AdipoRon (P=0.03 and 0.002, respectively), suggesting cell cycle delay or arrest (Figure 3A). Notably, in OVCAR4 cells treated with AdipoRon there was a trend toward G1 accumulation but was not significant. We next assessed MPM-2, a cell cycle marker that identifies a phospho-epitope unique to proteins involved in entry into mitosis. OVCAR3 and OVCAR4 cells were synchronized using a double thymidine block, then released and treated with AdipoRon for 24 hrs. Both OVCAR3 and OVCAR4 cells demonstrated decreased expression of MPM-2 when treated with AdipoRon, providing further evidence that AdipoRon is inhibiting mitotic progression (Figure 3B).

Figure 3: Effect of AdipoRon on cell cycle progression.

Figure 3:

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A) OVCAR3, OVCAR4 and A2780 ovarian cancer cells were treated with vehicle control or 50 μM AdipoRon for 24 hours before being fixed and stained with propidium iodide. Number of cells in each phase of the cell cycle were measured using flow cytometry. Analysis of relative cell population in each phase of the cycle are shown. B) OVCAR3 and OVCAR4 cells were synchronized via thymidine block and released and treated with 50 μM AdipoRon for 24 hours. Protein lysates were generated and used for immunoblot against MPM-2. Loading control = β-actin. Mean ± s.e.m., n=3, NS=not significant, **p<0.001, ***p<0.0001 vs. vehicle control.

AdipoRon induces apoptosis in ovarian cancer cells

To elucidate the potential pro-apoptotic effect of AdipoRon in ovarian cancer cells, we utilized both Annexin V and caspase-3 activation assays, which are independent methods to assess programmed cell death. In both assays, OVCAR3 and OVCAR4 ovarian cancer cells were treated with vehicle or 50 μM AdipoRon. Following treatment, ovarian cancer cells were either utilized for immunofluorescence against a classic apoptotic marker (cleaved caspase 3) or for flow cytometry to identify cells exhibiting Annexin V on the outer plasma membrane (a marker of cellular apoptosis). In both cells lines we observed an increase (P<0.001 for OVCAR3 and P=0.045 for OVCAR4) in cleaved caspase 3 positive cells when treated with AdipoRon compared to vehicle treated cells (Figure 4A). Utilizing flow cytometry, we also demonstrated that there were significantly more cells (P<0.001 for OVCAR3 and OVCAR4) with Annexin V on the cell surface in the population of cells treated with AdipoRon compared to vehicle (Figure 3B). Taken together, AdipoRon treatment is promoting apoptosis in ovarian cancer cell lines.

Figure 4: Effect of AdipoRon on apoptosis and cell number.

Figure 4:

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A) OVCAR3 and OVCAR4 ovarian cancer cells were treated with vehicle control or 50 μM AdipoRon for 24 hours. Cells were fixed and treated with antibody against cleaved caspase-3 and a nuclear stain (dapi) for immunofluorescence analysis of cell population positive for active caspase-3. B) Another set of cells were collected and stained with fluorescently labeled Annexin V and propidium iodide. Percent of the cell population positive for Annexin V is shown. C) OVCAR3, OVCAR4 and A2780 ovarian cancer cells were treated with vehicle control (white bars), 10 μM AdipoRon (gray bars) or 50 μM (black bars) AdipoRon for 24 hours. Cell number was determined measuring total DNA content and plotted as % of cell population present at 24 hours. Mean ± s.e.m., n=3, NS=not significant, *p<0.05, **p<0.001, ***p<0.0001 vs. vehicle control.

AdipoRon has an anti-proliferative effect on serous ovarian carcinoma in vitro

We next assessed whether AdipoRon reduced proliferation in ovarian cancer cell lines. We treated the OVCAR3 high grade serous ovarian carcinoma cell lines with varying doses of AdipoRon and measured the DNA content as surrogate for proliferation. We evaluated doses of AdipoRon ranging from 1 μM to 50 μM. After 48 hours of treatment, AdipoRon decreased OVCAR3 proliferation in a dose dependent manner (Supplemental Figure 1). We proceeded to treat OVCAR4 and A2780 high grade serous ovarian carcinoma cell lines with AdipoRon at doses of 10 μM and 50 μM (Figure. 4C). The 10 μM dose significantly decreased proliferation in the OVCAR3 (P=0.002) and OVCAR4 (P<0.001) lines, but not the A2780 cell line. The 50 μM dose significantly decreased cell proliferation in all cell lines (P<0.001 for all three cell lines). These data demonstrate that AdipoRon treatment inhibits ovarian cancer cell proliferation.

AdipoRon induces apoptosis in high grade serous primary ovarian cancer tumors

We next tested the effect of AdipoRon on primary patient tumors. Patient tumor sample slices in ex vivo culture, from the University of Colorado GTFB, were treated with vehicle or 50 μM AdipoRon. All three tumors samples tested were of high-grade serous histology. Each demonstrated a significant increase (P values ranging from <0.001 to 0.02) in cleaved caspase 3 in response to AdipoRon treatment compared to vehicle treatment (Figure 5). These findings suggest that the anti-proliferative effect of AdipoRon is relevant to human patient tumors, specifically ovarian tumors with the high grade serous histologic subtype.

Figure 5: Effect of AdipoRon on human tumor tissue.

Figure 5:

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Patient tumor samples were sliced and placed in culture. Tissue slices were treated with vehicle control or 50 μM AdipoRon for 24 hours. Slices were fixed, paraffin embedded and sectioned for H&E analysis or immunohistochemical analysis of cleaved caspase 3. Representative H&E and corresponding cleaved caspase-3 staining are shown (A). Quantification of caspase-3 staining from three individual patient tumors (B-D). Mean ± s.e.m., n=3 slices per treatment group, *p<0.05, ***p<0.0001 vs. vehicle control.

DISCUSSION

We are the first to report that the AdipoR1 and AdipoR2 synthetic agonist, AdipoRon, exerts an anti-proliferative and pro-apoptotic effect on high grade serous ovarian carcinoma cells. We demonstrate that AdipoRon activates AMPK, disrupts the cell cycle, and activates caspase-3 (Figure 6). These results suggest that AdipoRon can activate the same signaling pathways as exogenous adiponectin and these signaling pathways play an important role in regulating tumor cell survival.

Figure 6: Proposed model of AdipoRon activity in ovarian cancer.

Figure 6:

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Adiponectin is downregulated and TP53 (p53, blue) is commonly mutated in ovarian cancers. AdipoRon treatment activated AMPK, inhibited cell cycle progression, and promoted apoptosis. Dotted lines indicate future work.

Both AdipoR1 and AdipoR2 have been reported to be expressed in serous ovarian cancer cells [21]. Examination of adiponectin receptor expression in The Cancer Genome Atlas reveals that higher ADIPOR1 expression in ovarian tumors correlates with better overall survival for these patients, highlighting the importance of this receptor in ovarian cancer [22]. In cultured endometrial and colon cancer cell lines, the anti-proliferative effect of adiponectin appears to be mediated by both AdipoR1 and AdipoR2 [6, 7]. It remains to be determined whether the same is true for ovarian cancer cells. In one of the three cell lines (OVCAR4) AdipoRon promoted only a modest induction in AMPK phosphorylation and cell cycle arrest suggesting differential signaling between cell lines. For instance, OVCAR3 cells are known to have a cyclin E1 (CCNE1) amplification suggesting a hypersensitivity to cell cycle perturbation [23]. Further investigation into AdipoR1 and R2 expression as well as gain and loss of function studies in these cell lines are needed to clarify the roles of the receptors and their downstream signaling in ovarian cancer.

It is established that adiponectin activates AMPK in multiple tumor sites [19]. Stimulation of adiponectin receptors with exogenous adiponectin has been reported to inhibit proliferation in colon and endometrial cancer cells through activation of AMPK [6, 7]. In general agreement with these findings, our data suggest that AdipoRon activates AMPK signaling in ovarian cancer cells. These results are consistent with the possibility that the reduced AMPK activity typically observed in epithelial ovarian tumor cells is related to decreased circulating levels of adiponectin observed in patients with ovarian cancer [24] and that restoring adiponectin signaling by AdipoRon could reverse these pro-tumorigenic signaling pathways. We hypothesized that activation of AMPK inhibits the mTORC1 pathway in ovarian cancer cells, similar to what has been observed in breast cancer cells treated with adiponectin [25]. Inhibition of mTORC1 is associated with an increase in cell death in ovarian carcinoma [26]. Crosstalk between AMPK and mTOR signaling is established in “normal” cells [27]. We assessed mTORC1 signaling activity by determining the expression and phosphorylation of multiple mTORC1 downstream effectors and did not observe a significant change in OVCAR3 and OVCAR4 cells treated with AdipoRon. This finding suggests that a mTOR-independent pathway downstream of AMPK is driving the cell cycle arrest, anti-proliferative, and pro-apoptotic effects of AdipoRon. For instance, in glioblastoma AMPK activates Peroxisome Proliferator-Activated Receptor gamma to induce cell cycle arrest and apoptosis, in a similar fashion as observed in the ovarian cancer cell lines [28]. Also, the AdipoRon induced cell cycle arrest observed in ovarian cancer cells could be due to AMPK-dependent downregulation of cyclin D1, which has been reported in breast cancer models [29].

Adiponectin receptor activates multiple AMPK-independent signaling pathways including, but not limited to: mitogen activating protein kinase (MAPK), phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt), activator of transcription 3 (STAT3), c-Jun N-terminal kinase (cJNK), and Wnt signaling [30–32]. Many of these signaling pathways play important roles in cancer cell biology to promote growth and survival. The PI3K/Akt pathway has been shown to be directly inhibited by adiponectin in colorectal cancer, which also leads to downstream decrease in mTOR activity [33]. Adiponectin treatment of breast cancer cell lines resulted in downregulation of the tumorigenic Wnt/beta-catenin signaling pathways via stimulation of Wnt inhibitory factor-1 [34, 35]. Determining whether AdipoRon regulates these pathways in ovarian tumor cells is an important next step in understanding the role of adiponectin receptor signaling in ovarian cancer.

The mechanistic underpinnings that link obesity, adiponectin and ovarian cancer remain to be fully established. Adiponectin is primarily secreted by adipocytes of white adipose tissue and circulating levels of the hormone are inversely related to body fat mass. Ovarian cancer is associated with low adiponectin also in patient with normal BMIs, suggesting that something other than adiposity may be regulating adiponectin secretion in this patient population [11]. The omentum, which is a common site of ovarian cancer metastatic implants, is a significant source of circulating adiponectin [36, 37]. It is possible that impaired secretion of adiponectin from a diseased and dysfunctional omental tissue contributes to the lower circulating levels of adiponectin in ovarian cancer patients, independent of obesity, and may serve as a diagnostic marker. Results from this study suggest that AdipoRon could potentially restore adiponectin signaling to halt tumor growth.

AdipoRon was originally described as a strategy to restore metabolic homeostasis in a mouse model of diabetes [13]. Messaggio and colleagues recently reported that treatment of pancreatic tumors with AdipoRon inhibited proliferation, promoted apoptosis, and reduced tumor size [38]. Our study demonstrates AdipoRon activates AMPK in cultured ovarian cancer cells and induces apoptosis in both cultured and primary tumor specimens. Therefore, AdipoRon treatment presents the exciting possibility of being both a preventative and a therapeutic strategy for ovarian cancer.

Supplementary Material

1

Acknowledgements

This research was supported in part by University of Colorado Department of Obstetrics and Gynecology Academic Enrichment Fund grant. The authors appreciate the contribution to this research made by E. Erin Smith, HTL(ASCP)CM QIHC, Allison Quador, HTL(ASCP)CM and Jessica Arnold HTL(ASCP)CM of the University of Colorado Denver Tissue Biobanking and Histology Shared Resource. This resource is supported in part by the Cancer Center Support Grant (P30CA046934). Flow cytometry was performed by the University of Colorado Cancer Center Flow Cytometry Shared Resource (supported by NCI Cancer Center Support Grant P30CA046934). We would also like to acknowledge that imaging was made possible by the University of Colorado Advanced Light Microscopy Core, supported in part by NIH/NCRR CCTSI grant UL1 RR025780. Contents are the authors’ sole responsibility.

Footnotes

Conflict of Interest Statement: All authors declare that they have no conflicts of interest related to this work.

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