Adipocyte-induced CD36 expression drives ovarian cancer progression and metastasis

Abstract

Ovarian cancer (OvCa) is characterized by widespread and rapid metastasis in the peritoneal cavity. Visceral adipocytes promote this process by providing fatty acids (FAs) for tumour growth. However, the exact mechanism of FA transfer from adipocytes to cancer cells remains unknown. This study shows that OvCa cells co-cultured with primary human omental adipocytes express high levels of the FA receptor, CD36, in the plasma membrane, thereby facilitating exogenous FA uptake. Depriving OvCa cells of adipocyte-derived FAs using CD36 inhibitors and short hairpin RNA knockdown prevented development of the adipocyte-induced malignant phenotype. Specifically, inhibition of CD36 attenuated adipocyte-induced cholesterol and lipid droplet accumulation and reduced intracellular reactive oxygen species (ROS) content. Metabolic analysis suggested that CD36 plays an essential role in the bioenergetic adaptation of OvCa cells in the adipocyte-rich microenvironment and governs their metabolic plasticity. Furthermore, the absence of CD36 affected cellular processes that play a causal role in peritoneal dissemination, including adhesion, invasion, migration and anchorage independent growth. Intraperitoneal injection of CD36-deficient cells or treatment with an anti-CD36 monoclonal antibody reduced tumour burden in mouse xenografts. Moreover, a matched cohort of primary and metastatic human ovarian tumours showed upregulation of CD36 in the metastatic tissues, a finding confirmed in three public gene expression data sets. These results suggest that omental adipocytes reprogram tumour metabolism through the upregulation of CD36 in OvCa cells. Targeting the stromal-tumour metabolic interface via CD36 inhibition may prove to be an effective treatment strategy against OvCa metastasis.

Figure 1. Adipocytes induce CD36 expression in ovarian cancer cells

(a) Western blot analysis of CD36 protein expression in OvCa cell lines co-cultured with human primary adipocytes (HPA) at the indicated time points. (b) Western blot analysis of FA transport proteins FABPpm, FATP1 and FATP4 in three different OvCa cell lines co-cultured with HPA for 24 h. (c) Confocal immunofluorescent microscopy for the indicated fatty acid transporters in the absence (-) and presence (+) of HPA. Fatty acid transporters are detected using green fluorescence, plasma membrane using red fluorescence (Wheat Germ Agglutinin), cell nuclei are counterstained with Hoechst 33258 (blue). (d) Subcellular localization of CD36 protein. OvCa cells were either co-cultured with HPA or were grown in serum free media (SFM). After 16 h, cells were either lysed to obtain total cellular lysate (TCL) or fractionated to cytoplasmic (CP) and plasma membrane (PM) fractions.

Figure 2. Inhibition of CD36 reduces adipocyte induced fatty acid uptake and lipid accumulation

(a) Confocal immunofluorescence microscopy to detect CD36. SKOV3ip1 cells were cultured alone or with HPA for the indicated times and fixed on glass coverslips followed by incubation with a CD36 primary antibody and an Alexa-488 conjugated secondary antibody (green). Nuclei were counterstained with Hoechst 33258 (blue). Scale bars, 10 μm. (b and c) Effects of adipocyte conditioned media (CM) and the CD36 specific inhibitor Sulfo-N-Succinimidyl Oleate (SSO) on parental (SKOV3ip1), scramble control (SKOV3ip1 shRNA-Scramble) and CD36 shRNA transduced cells (SKOV3ip1 shRNA-CD36) over time. Cancer cells (2.5×10⁵) were serum-starved and when indicated incubated with adipocyte-CM and/or the irreversible CD36 inhibitor, SSO. FA transport was measured with a fluorescently labeled fatty acid (BODIPY-FA) and a nontoxic cell-impermeable quenching agent (Q-Red.1) at the indicated time points. Exclusion of the quenching reagent from the cells results in an increase in fluorescent signal intensity originating from intracellular BODIPY-FA. Changes in intracellular FA levels are quantified with a fluorescent plate reader as described in the materials and methods. ***p<0.001. (d) Intracellular lipid accumulation in parental (SKOV3ip1), scramble control (SKOV3ip1 shRNA-Scramble) and CD36 shRNA transduced cells (SKOV3ip1 shRNA-CD36) cultured with HPA. After 16 hrs. of co-culturing, neutral lipid droplets (LD) were stained with BODIPY 493/503 and total LD area was calculated with Image J. Bars represent the mean ± s.e.m. from 3 independent experiments (n.s., not significant, **p<0.01) Representative immunofluorescent images of cellular LD accumulation (LD in green, BODIPY 493/503 and nuclei in blue, Hoechst 33258) are shown.

Figure 3. Microarray analysis of ovarian cancer cells with adipocyte co-culturing

(a) Canonical pathways altered by human primary adipocyte (HPA) co-culturing. Pathways are ranked based on their significance (p-value) calculated using the right-tailed Fisher’s exact test. The p-value for each pathway is indicated by the bar and is expressed as -1 times the log of the p-value. (b) Ingenuity Network Analysis. Network 8, changes in lipid metabolism in response to HPA stimulation. Gene products are represented as nodes and biological relationships between two nodes as a line. Red and green indicate up- and down- regulation, respectively. The color intensity correlates with the degree of change. Genes in white did not exhibit significant changes in expression.

Figure 4. CD36 depletion affects cellular metabolism

(a) Western blot analysis of total AMP-activated protein kinase (total AMPKα) and phospho-AMPK (p-AMPKα) levels in scramble control and CD36 shRNA transduced cells (CD36 shRNA) in the absence (-) and presence (+) of human primary adipocytes (HPA) at the indicated time points. The p-AMPK antibody recognizes T172 in the catalytic subunit of AMPK. (b) Glycolytic function. Scramble control (SKOV3ip1 shRNA-Scramble) and CD36 shRNA transduced cells (SKOV3ip1 shRNA-CD36) were incubated with or without adipocyte conditioned media (CM). Extracellular acidification (ECAR) rate was measured with the Seahorse XF24 Analyzer. Data are means ± s.e.m. of seven replicates. (c) Basal oxygen consumption rate. Scramble control (SKOV3ip1 shRNA-Scramble) and CD36 shRNA transduced cells (SKOV3ip1 shRNA-CD36) were incubated with or without adipocyte conditioned media (CM). OCR, oxidative phosphorylation and ATP production were measured with the Seahorse XF24 Analyzer. Data are means ± s.e.m. of four replicates. (d) Cholesterol assay. Intracellular cholesterol accumulation was measured by the cholesterol oxidation reaction method using fluorescence in the absence and presence (+ HPA) of human primary adipocytes. All data shown are means, ± s.e.m. (n=3) **p<0.01, n.s., not significant. Data are representative of three independent experiments. (e) TBAR assay. Oxidized lipid production was quantified with fluorometric measurement of malondialdehyde (MDA) formation in the absence and presence (+ HPA) of human primary adipocytes. All data shown are means, ± s.e.m. (n=3) **p<0.01, *p<0.05. Data are representative of three independent experiments.

Figure 5. Silencing of CD36 impairs invasion, migration, adhesion and clonogenic capacity of SKOV3ip1 cells in vitro

(a) Invasion (left) and migration (right) of parental (SKOV3ip1), scramble vector (SKOV3IP1 shRNA-Scramble) and CD36 shRNA-1 transduced (SKOV3ip1 shRNA-CD36) cells towards, serum free media (SFM) or primary human adipocytes (HPA). Invaded and migrated cells were counted with Image-J. Data are representative of three independent experiments performed in triplicates. ***p<0.001. (b) Adhesion of SKOV3ip1 shRNA-CD36 cells to the indicated extracellular matrix proteins. Fluorescently labeled SKOV3ip1 cells were plated onto 96-well plates coated with the indicated ECM components. Plates were washed and the number of adherent cells were quantified by measuring fluorescence intensity and a standard curve. Percent change in adhesion of CD36 shRNA transduced cells compared to scrambled vector and normalized to poly-d-lysine coated wells. Bars represent the means ± s.e.m. from two independent experiments (n=5). **p<0.01. (c) Colony formation. SKOv3ip1 cells (4,000) were plated onto soft agar; after 33 days, colonies were stained with crystal violet and counted. The data are representative of three independent experiments and columns represent the mean from three different wells. **p<0.01, n.s., not significant. Representative images of stained colonies are shown.

Figure 6. CD36 inhibition reduces in vivo tumour growth

Xenograft models of metastasis. Female athymic mice were injected i.p with (a) 1×10⁶ scrambled vector or CD36 shRNA transduced SKOV3ip1cells and were sacrificed after 32 days or (b) 5×10⁶ OVCAR-8 cells. One week following tumour inoculation, animals were treated for 4 weeks with a daily injection of anti-CD36 monoclonal antibody or its isotype control. At the conclusion of the experiments, tumours were counted, excised, and weighed. The number of tumour metastases (top) and tumour weight (bottom) were determined. Each point represents an individual animal and the horizontal bar is the mean (***p<0.001, **p<0.01). Representative images of omental and peritoneal tumour nodules are shown from four different animals. Arrows show mesenteric tumour nodules, dashed lines highlight omental and peritoneal metastases.

Figure 7. Relative CD36 mRNA levels are increased in ovarian cancer metastasis

(a) Scatter diagram showing quantitative Real-time PCR comparison of relative CD36mRNA levels from laser micro dissected high grade serous human ovarian tumours and their corresponding omental metastases (n=10). The log ratio of paired metastatic and primary OvCa samples is plotted, >1 is higher in metastasis (n=7) and <1 is lower in metastasis (n=3). p<0.05, obtained by paired Wilcoxon Signed Ranks test. (b) Oncomine analysis of CD36 expression in primary and metastatic high grade serous ovarian carcinomas. Oncomine datasets containing primary tumours as well as omental metastasis (top row) or peritoneal metastasis (bottom row) were queried for CD36 expression. Box legend: + inside box represents mean value, bar inside box represents median value, upper bar represents maximum of distribution, p-values and fold changes are indicated below the x-axis of each graph. All p-values represent a student’s t-test. Data are publicly available on Oncomine, and citations are included in the Supplementary Data.