Overexpression of CD36, Driven By STAT3, Mediates Free Fatty Acid Uptake in CLL Cells

Unlike their quiescent normal B-cell counterparts, CLL cells proliferate and in most patients they multiply by approximately 1% daily. We have recently found that to adjust for the increase in the cells' energy demand, CLL cells undergo a metabolic reprogramming. Like adipocytes that proliferate at a similar rate, CLL cells store lipids in their cytosol and utilize free fatty acids (FFA) as an energy source. However the cellular pathways that participate in this process and how they affect the metabolic adaptation of CLL cells has not been fully elucidated.

CD36, a member of the B scavenger receptor family, is a protein that is expressed on the surface of many cell types and is known to play a role in lipid metabolism. In adipocytes CD36 binds and internalizes lipoproteins and long-chain FFA. In line with other reports, a recent study showed that in CD36-null mice adipocytes' lipid uptake is severely impaired, confirming that CD36 is required for FFA uptake in mouse adipocytes.

The signal transducer and activator of transcription (STAT)-3 is ubiquitously expressed in mammalian cells. Upon phosphorylation, STAT3 forms dimers, shuttles to the nucleus, binds to DNA, and functions as a transcription factor. Because STAT3 is constitutively phosphorylated in CLL cells, and because we found that the CD36 gene promoter harbors several putative STAT3 binding sites, we hypothesized that STAT3 induces the expression of CD36 and that CD36 contributes to the metabolic reprogramming of CLL cells.

Using Western immunoblotting we quantified the levels of CD36 in 6 CLL patients and found that in CLL cells from all patients, CD36 levels were significantly higher than in CD19+ normal B cells. Furthermore, using flow cytometry we found that CD36 was located on the surface of CD5+/CD19+ CLL cells. In addition, by using confocal microscopy we confirmed that CD36 is present on the surface but not in the cytoplasm or nucleus of CLL cells. Sequence analysis identified 4 putative STAT3 binding sites 200 bp upstream of the CD36 gene start codon. To determine whether STAT3 binds to the CD36 promoter we first used chromatin immunoprecipitation (ChIP) and found that STAT3 binds to 3 of the 4 putative STAT3 binding sites. Then, using an electromobility shift assay (EMSA) we confirmed that STAT3 binds to the same 3 putative binding sites but not to the site that did not co-immimmunoprecipitate with STAT3 in the ChIP assay. To further elucidate the role of CD36 in CLL cells, we transfected CLL cells with STAT3-small interfering (si)RNA and found that STAT3-siRNA downregulated CD36 expression by 7-fold and significantly reduced CD36 protein levels, suggesting that STAT3 induces CD36 expression and protein production in CLL cells. To test whether CD36-mediated uptake of FFA contributes to lipid metabolism in CLL we incubated CLL cells from 3 different patients in the presence and absence of FFA and measured the dissolved O₂ (dO₂) in the cells' culture medium. In all cultures the levels of dO₂ were markedly reduced in the presence, but not in the absence, of FFA, suggesting that CLL cells metabolized FFA. However, when CLL cells were transfected with CD36-siRNA or incubated with CD36 neutralizing antibodies, dO₂ levels did not significantly decrease, suggesting that the transfected cells or the cells that were incubated with CD36-siRNA could no longer utilize FFA. Finally, we incubated CLL cells with sulfosuccinimidyl oleate sodium (SSO), an inhibitor of CD36-mediated FFA uptake, or orlistat, an inhibitor of lipoprotein lipase. We found that SSO or orlistat reduced CLL cells' capacity to metabolize FFA and induced apoptosis of CLL cells in a dose dependent manner.

In conclusion: CLL cells adjust their metabolic program to meet an increased metabolic demand. Overexpression of CD36 enables cellular uptake of FFA. This process is driven by STAT3 which is constitutively phosphorylated in CLL cells and activates the expression of CD36. Hence, inhibiting FFA uptake by using SSO, currently in clinical use for weight loss, or other agents that inhibit the consumption of FFA by CLL cells might prove to be a potential novel therapeutic approach in CLL.