Background
Pancreatic cancer is the fourth most common cause of cancer mortality and recognized as the “king of cancers” in the world [
1,
2]. This deadly disease is dependent on mitochondrial function for enhanced survival and aggressiveness, which is extremely difficult to detect in the early stages because of frequently few symptoms and lacking effective diagnosis. Despite significant improvements in clinical managements over the past two decades, the 5-year survival rate for pancreatic cancer patients remains lower than 10% [
1‐
4]. Currently, the medical treatments are FOLFIRINOX (a four-drug combination of fluorouracil, leucovorin, irinotecan, and oxaliplatin) and gemcitabine plus nab-paclitaxel (G-nab), which provide a median overall survival of 11.1 months and 8.5 months, respectively [
5,
6]. However, these treatments have moderately toxic effects and are often used to treat pancreatic cancer patients with good performance status. Therefore, safe and effective anticancer drugs are urgently needed in order to significantly prolong patients’ survival.
Lipoic acids, are mostly synthesized within the mitochondria as a cofactor necessary during mitochondrial energy metabolism, which have been shown to decrease cell viability and proliferation in pancreatic, breast, colon, ovarian, and lung cancer cells [
7,
8]. CPI-613 (Devimistat) is the first member of a large set of analogs of lipoic acids, which strongly induces tumor repression by changing mitochondrial enzyme activity and redox status [
9,
10]. CPI-613 is used as an inhibitor of mitochondrial tricarboxylic acid (TCA) for cancer treatment, because it can specifically target pyruvate dehydrogenase (PDH) and alpha-ketoglutarate dehydrogenase (α-KGDH) involved in the TCA cycle [
11,
12]. The anticancer activity of CPI-613 has been confirmed against human pancreatic cancer in xenograft models with low side-effect toxicity [
13]. A Phase I study reported there was a 61% objective response rate (including a 17% complete response rate) for metastatic pancreatic cancer patients receiving combination of CPI-613 with modified FOLFIRINOX (mFFX) [
10]. A Phase III open-label trial to evaluate efficacy and safety of CPI-613 combined with mFFX versus FFX in patients with metastatic pancreatic cancer are now undergoing [
14]. Nevertheless, the underlying molecular mechanisms of CPI-613 remain to be determined.
In this study, we show for the first time that the 5′ AMP-activated protein kinase (AMPK)-Acetyl-coenzyme A carboxylase (ACC) signaling is deeply involved in CPI-613-induced apoptosis in pancreatic cancer. Mechanistically, CPI-613 activates AMPK in pancreatic cancer cells, which in turn triggers autophagy and ACC inhibition. Interestingly, autophagy only marginally affects CPI-613-induced apoptosis. It appears that AMPK-dependent ACC inhibition contributes to reduced lipid metabolism upon CPI-613, which augments reactive oxygen species (ROS)-associated apoptosis in pancreatic cancer cells. These observations reveal that CPI-613 rewires lipid metabolism to enhance pancreatic cancer apoptosis via the AMPK-ACC signaling, providing new insights into the crosstalk between lipid metabolism reprogramming and apoptosis in cancer treatment.
Methods
Cell lines and culture
Human pancreatic cancer cell lines AsPC-1 and PANC-1 were purchased from the American Type Culture Collection (ATCC, Rockville, MD), and cultured in RPMI1640 medium containing 10% fetal bovine serum (FBS) at 37 °C in a humidified incubator supplied with 5% CO2.
Reagents, antibodies, and standard assays
CPI-613 was obtained from Selleckchem (Houston, TX). 2′,7′-dichlorofluorescin diacetate (DCFH-DA), chloroquine (CQ), N-acetylcysteine (NAC), Compound C, simvastatin, and 5-(tetradecyloxy)-2-furoic acid (TOFA) were purchased from Sigma-Aldrich (St Louis, MO). Fatty acid and lipid metabolism antibody sampler kit and glycolysis antibody sampler kit were purchased from Cell Signaling Technology (Beverly, MA). Fatty acid and lipid metabolism antibody sampler kit includes antibodies against Acetyl-CoA Carboxylase (ACC), p-Acetyl-CoA Carboxylase (p-ACC) (Ser79), AceCS1, ACSL1, Lipin 1, ATP-Citrate Lyase, p-ATP-Citrate Lyase, Fatty Acid Synthase (FAS). Glycolysis antibody sampler kit includes GAPDH, PDH, HKI, HKII, LDHA, PKM2, PKM1/2, and PFKP. Antibodies against c-Caspase 3, PARP, p-mTOR (Ser2448), mTOR, p62, LC3B, p-AMPKα (Thr172), AMPKα, p-ULK1, ULK1, Bax and Bcl-2 were purchased from Cell Signaling. Apoptotic rate was determined using Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, San Jose, CA). All flow cytometry data was analyzed using FlowJo software (Tree Star, Ashland, OR). Cell viability was determined by MTT assay, crystal violet staining, and alamarBlue Cell Viability Reagent (Thermo Fisher Scientific, Waltham, MA). Western blot, plasmid transfection and lentiviral infection were carried out as we previously described [
15‐
17].
Gene knockdown
Lentiviral-shRNA against ULK1 was obtained from GeneCopoeia (Rockville, MD), and the stable ULK1 knockdown AsPC-1 cells were generated using Lenti-Pac™ FIV Expression Packaging Kit (GeneCopoeia) according to the manufacturer’s instructions. Non-target shRNA (shNC) was used as a negative control in this study and the knockdown effect was confirmed by Western blot.
JC-1 analysis for mitochondrial membrane potential (MMP)
MMP was measured by the JC-1 fluorescent probe (Invitrogen, Carlsbad, CA). CPI-613-treated or non-treated cells were incubated with JC-1 (1:1000 dilution) for 20 min at 37 °C. After PBS washing, cells were observed under a fluorescence microscope with the red fluorescence (550 nm excitation/600 nm emission) and green fluorescence channels (485 nm excitation/535 nm emission). Quantitative analysis of Red/Green fluorescence ratio was measured by NIH ImageJ software.
Measurement of ROS levels
Intracellular ROS production was determined using the oxidant-sensing fluorescent probe DCFH-DA. Briefly, cells were incubated with 10 μM of DCFH-DA for 20 min at 37 °C and images were captured using a fluorescence microscope (IX-71, Olympus Corp., Tokyo, Japan). Median fluorescence intensity from at least 100 cells in randomly selected fields were quantified by NIH Image J software as we previously described [
18,
19].
Confocal laser scanning by high content analysis
The tandem labeled mRFP-GFP-LC3B plasmid was purchased from GeneCopoeia (Rockville, MD). In brief, cells were seeded into a 6-well plate overnight and transiently transfected with mRFP-GFP-LC3B using Lipofectamine 2000 according to the manufacturer’s instructions. After 48 h of transfection, cells were reseeded in CellCarrier 96-well microplates (PerkinElmer) in the presence or absence of 200 μM CPI-613. After a 24-h treatment, the fluorescent autophagy marker mRFP-GFP-LC3B was observed using a confocal laser mode (PerkinElmer, USA). The average number of mRFP-GFP-LC3B dots per cell was determined from three independent experiments. At least ten random fields representing 200 cells were counted in each well.
Transmission electron microscopy (TEM)
Approximately 1.0 × 107 cells treated with 200 μM CPI-613 or vehicle were fixed with 2% glutaraldehyde in 0.1 M sodium cacodylate (NaCAC) buffer (pH 7.4) for 45 min. The samples were post-fixed in 2% osmium tetroxide in NaCAC, stained with 2% uranyl acetate, dehydrated with a graded ethanol series and embedded in Epon-Araldite resin. Thin sections were cut with a Leica EM UC6 ultramicrotome (Leica Microsystems), collected on copper grids, and stained with uranyl acetate and lead citrate. Cells were observed in a Hitachi HT7700 transmission electron microscope and imaged with an UltraScan 4000 CCD camera and First Light Digital Camera Controller (Gatan).
Three-dimensional (3D) cell culture
Briefly, 1 × 10
5 cells were seeded into 48-well SeedEZ scaffold (Lena Bioscience, Atlanta, GA) supplied with complete medium. After 3 days of culture, cells growing in the SeedEZ scaffold were treated with 200 μM CPI-613 for 5 days, and cell viability was measured by alamarBlue at 545/590 nm ex/em, followed by phalloidin staining and imaging as we previously described [
20].
Lipolysis analysis
Lipid droplets and free fatty acids (FFA) released into the culture medium of pancreatic cancer cells were measured to evaluate lipolysis. AsPC-1 and PANC-1 cells were treated with 200 μM CPI-613 for 48 h prior to lipolysis assessment. To determine lipid droplets, cells were fixed with 4% paraformaldehyde and stained with the dye Oil-Red-O (Sigma-Aldrich) for 30 min using the isopropanol method, followed by processed for haematoxylin staining. The released FFA levels were measured by Free Fatty Acid Quantification Kit (Abcam, Cambridge, UK) according to the manufacturer’s instruction. The absorbance at 570 nm was measured immediately afterwards on a microplate reader.
Statistical analysis
Statistical analyses were performed by unpaired Student’s t test for two group comparisons and one-way analysis of variance (ANOVA) for multi-group comparisons at a significance level of p < 0.05. Data were presented as means ± SD from three or more independent experiments.
Discussion
Pancreatic cancer has an exceedingly poor prognosis with a 5-year survival compared with many other solid tumors [
1‐
4]. Current strategies do not target genetic features of pancreatic cancer and patients with this type of cancer have few therapeutic options [
2‐
4]. CPI-613, a novel lipoate analog with the function inhibiting mitochondrial metabolism, has been reported to produce strong tumor growth inhibition with little or no side-effect toxicity at 25 mg/kg or even higher therapeutic doses [
9]. Further clinical Phase I study reveals the maximum tolerated dose of CPI-613 was 500 mg/m
2 in pancreatic cancer patients enrolled between 2013 and 2016 [
10]. The median number of treatment cycles given at the maximum tolerated dose was 11, and the median follow-up of patients treated at the maximum tolerated dose was 378 days [
10]. This study also provides encouraging evidence that the novel treatment modality of CPI-613 in combination with mFFX chemotherapy was safe and well tolerated in patients with metastatic pancreatic cancer [
10]. Although a Phase III clinical trial of this treatment has been designed and is undergoing [
14], the underlying molecular mechanism remains unknown. We report here that CPI-613 exhibits strong anticancer activity in pancreatic cancer cells via ROS-associated apoptosis, which is coupled with AMPK activation. Upon CPI-613, the upregulated AMPK-ACC signaling rewires lipid metabolism, promoting the progression of apoptosis in pancreatic cancer cells. This novel mechanism explores the critical role of AMPK-dependent ACC inhibition in CPI-613-induced apoptosis, adding a new layer to understand the crosstalk between lipid metabolism and apoptosis in cancer treatment (Fig.
7g). Thus, our study may shed light on CPI-613-based treatment to lead to desired effect in pancreatic cancer patients.
The TCA cycle-mediated generation of ROS is a key mediator for cell survival. CPI-613 has been reported to selectively target the altered form of mitochondrial TCA in tumor cells, leading to apoptosis via changes in mitochondrial enzyme activities and redox status [
11]. This is true in pancreatic cancer cells as we provide the evidence that CPI-613-induced apoptosis is tightly associated with ROS. Inhibiting ROS in these cells using the antioxidant NAC can prevent cell from apoptosis in CPI-613 treatment. Most interestingly, CPI-613-induced apoptosis is also regulated by the AMPK signaling. While mTOR and ULK1 are two well-reported targets of AMPK that can be co-activated by AMPK and subsequently trigger autophagy [
15,
22], our findings unravel that autophagy triggered by CPI-613 only lies in the AMPK-ULK1 signaling.
Autophagy is a self-protective response in living cells or organisms to adapt to stress and extracellular cues [
15,
22]. The mutual relationship between autophagy and apoptosis is highly context-dependent. A majority of cases, it seems that apoptosis and autophagy are mutually inhibitory, although there is accumulating evidence showing autophagy tends to be pro-apoptotic rather than anti-apoptotic in some particular conditions [
22,
27]. The observations from the present study suggest that AMPK-dependent autophagy only has a marginal effect on CPI-613-induced apoptosis in pancreatic cancer cells, which prompted us to study the other possible role of AMPK in CPI-613 treatment as it is considered to be an important component controlling many other pathways.
Lipid metabolism participates in the regulation of many cellular processes (e.g. cell survival and apoptosis) and its disorder is pathologically linked to cancer [
28]. ACC as the rate-limiting enzyme in fatty acid synthesis, plays a pivotal role in the growth and viability of cancer cells [
29]. Phosphorylation by AMPK converts ACC to an inactive form, leading to the reduction in lipid metabolism. In this study, we explore that CPI-613 is potent to increase the ACC phosphorylation levels through activating the AMPK signaling in pancreatic cancer cells. We also reveal that inactivation of either AMPK or ACC attenuates CPI-613-induced apoptosis, suggesting the deep involvement of the AMPK-ACC signaling in treatment-associated apoptosis. Although the results from pancreatic cancer cell lines are sufficient to prove the role and importance of AMPK-ACC signaling in CPI-613 treatment, the current study did not include the in vivo confirmation. It would be interesting to determine whether this molecular mechanism is highly relevant to the therapeutic outcomes of CPI-613 in preclinical cancer models. Moreover, whether ROS affects the AMPK-ACC signaling axis and how ACC-mediated lipid metabolism contributes to ROS-associated apoptosis, are warranted to better understand the drug action of CPI-613.
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