Lipoprotein lipase links dietary fat to solid tumor cell proliferation

Abstract

Many types of cancer cells require a supply of fatty acids (FA) for growth and survival, and interrupting de novo FA synthesis in model systems causes potent anticancer effects. We hypothesized that, in addition to synthesis, cancer cells may obtain preformed, diet-derived FA by uptake from the bloodstream. This would require hydrolytic release of FA from triglyceride in circulating lipoprotein particles by the secreted enzyme lipoprotein lipase (LPL), and the expression of CD36, the channel for cellular FA uptake. We find that selected breast cancer and sarcoma cells express and secrete active LPL, and all express CD36. We further show that LPL, in the presence of triglyceride-rich lipoproteins, accelerates the growth of these cells. Providing LPL to prostate cancer cells, which express low levels of the enzyme, did not augment growth, but did prevent the cytotoxic effect of FA synthesis inhibition. Moreover, LPL knockdown inhibited HeLa cell growth. In contrast to the cell lines, immunohistochemical analysis confirmed the presence of LPL and CD36 in the majority of breast, liposarcoma, and prostate tumor tissues examined (n = 181). These findings suggest that, in addition to de novo lipogenesis, cancer cells can use LPL and CD36 to acquire FA from the circulation by lipolysis, and this can fuel their growth. Interfering with dietary fat intake, lipolysis, and/or FA uptake will be necessary to target the requirement of cancer cells for FA.

Figure 1

LPL, CD36, and FASN gene expression in cancer cells. Ethidium-stained gel electrophoresis of RT-PCR products is shown in Panels A–C. Cell lines analyzed are listed above each lane. Stds indicates electrophoretic size standards; LiSa-2 is a liposarcoma line; Du4475 are breast cancer cells lacking receptors for sex steroids and trastuzumab; T47D are breast cancer cells with receptors for estrogen and progesterone, but not trastuzumab; BT474 are breast cancer cells with receptors for sex steroids and trastuzumab. PC3, LNCaP, and VCaP are prostate cancer lines; Fibro denotes human fibroblasts. Panel A: Primers corresponded to cyclophilin (cyclo) or LPL mRNAs. Panel B: Primers corresponded to the fatty acid translocase CD36. Panel C: Primers detected FASN mRNA. Panel D depicts real time RT-PCR quantitation of LPL mRNA (normalized to 18S rRNA, mean ± SEM, n = 3 wells/cell line). HeLa adenocarcinoma cells are included as a positive control, as we previously reported expression of LPL mRNA by this cell line (25).

Figure 2

Production of lipoprotein lipase activity by breast cancer, liposarcoma, and prostate cancer cells and in a breast cancer tissue sample. Panel A: Lipase activity is shown (mean ± SEM, 4 samples/group, corrected for cellular protein content and normalized to the value observed in milk (9 × 10³ cpm/2h). Human breast milk (50 μl), mouse gastrocnemius muscle (50 mcg protein, 45 × 10³ cpm/2h), or tissue culture media conditioned by the indicated cell lines x 3 d were assessed for lipase activity (mean ± SEM, 4 samples/group, corrected for cellular protein content and activity observed in unconditioned media). The dotted line denotes the LPL activity found in unconditioned culture medium. Panel B: Time course of accumulation of lipase activity in conditioned culture media. Media (50 μl) were removed from cultures at the indicated intervals (mean cpm/mg protein ± SEM, n = 4 wells/timepoint). Panel C, upper: Identification of LPL in conditioned cell culture media. LPL was heparin-sepharose affinity purified from 10 ml fresh culture medium, 1.0 ml human breast milk, or 10 ml culture media conditioned (72 h) by LiSa-2 liposarcoma or DU4475 “triple negative” breast cancer cells, eluted with 0.6–0.8 M NaCl, and analyzed by western blot using anti-human LPL clone 43 (1:200). The band from milk was verified to contain LPL by mass spectrometry. Panel C, lower: Western analysis of a breast tumor homogenate (50 mcg protein without affinity purification) and breast milk (10 μl) for LPL. A band of the appropriate size is apparent in the tumor sample. Panel D: Estimation of the heparin-releasable LPL pool in breast cancer tissue and HeLa cells. Panel D left: tumor associated LPL activity is significantly reduced by heparin treatment (p = 0.0001). Panel D: right: Heparin reduced LPL activity residing in HeLa cell pellets by 29% (p < 0.04).

Figure 3

LPL stimulates tumor cell growth in the presence of lipoproteins. Panel A: T47D breast cancer cells were grown x 72 h in media containing complete or lipoprotein-depleted fetal calf serum (triglyceride content 660 and 20 μg/ml, respectively) plus the indicated concentrations of LPL. Media were replaced at 24 h intervals. Data in this and other panels are mean ± SEM, normalized to the control group (seeded in 24 well plates at 20k cells/well, n = 6 wells/group). *p< 0.05 compared to control. Panel B: LiSa-2 liposarcoma cells were grown x 72 h in media containing complete or lipoprotein-depleted fetal calf serum plus the indicated concentrations of LPL were replaced at 24 h intervals. *p< 0.05 compared to control. Panel C: LnCaP prostate cancer cells were treated with the indicated concentrations of LPL +/− 100 nM Soraphen A to inhibit lipid synthesis. Comparisons are within the lipoprotein plus and minus groups. *p< 0.05 compared to no LPL or Soraphen A, #p< 0.05 compared to no LPL, + Soraphen A. Panel D: PC3 prostate cancer cells were treated as in Panel C. *p< 0.05 compared to no LPL or Soraphen A, #p< 0.05 compared to no LPL, + Soraphen A. Panel E: Two LPL siRNAs (A, B), but not a nonspecific siRNA (NS) cause a substantial decline in LPL mRNA. Data are mean LPL mRNA signal normalized to 18S RNA± SEM, 4 wells/group. RNA was harvested 48 h after transfection. *p<0.05 compared to the nonspecific siRNA. Panel F: LPL siRNA impairs the growth of HeLa cells, and augments the antiproliferative effect of Soraphen A. Data are viable cells/well, mean ± SEM (n = 4 wells/group). Cell growth was assessed 96 h after siRNA transfection. *p< 0.05 compared to the no siRNA, no Soraphen A, no LPL group, #p< 0.05 compared to the control siRNA, no Soraphen group, @p< 0.05 compared to the respective siRNA groups (A, B) without Soraphen A.

Figure 4

Immunohistochemical analysis of markers of fatty acid metabolism in breast, liposarcoma, and prostate tumors. Slides from a representative invasive ductal carcinoma of the breast (left column), liposarcoma (middle column), and prostatic adenocarcinoma (right column) were immunostained for A: fatty acid synthase (FASN), B: THRSP (Spot 14, S14), C: lipoprotein lipase (LPL), or D: CD36. Original magnification was 40x. Detection was with peroxidase (brown pigment), and slides were counterstained with hematoxylin (blue pigment). FASN staining is cytosolic, and S14 is nuclear. LPL demonstrated an asymmetric, perinuclear distribution (arrows in insets) compatible with localization to the Golgi apparatus. Note that the well-visualized prostate tumor stroma does not express detectable LPL. CD36 exhibited two distinct patterns of subcellular localization in breast tumors. Only a cytosolic signal was seen in 29% of cases (row D, left, upper panel), whereas prominent cell surface staining was seen in 69% (lower panel), and only 2% were devoid of CD36 immunoreactivity. Staining was also seen in most liposarcoma and prostate cancers. This was primarily a cell surface pattern, and was not uniformly present across those tumors.