We used a cDNA microarray to screen 45 breast cancer-derived cell lines from the dataset of Neve and coworkers (11
) for LPL gene expression, and for FASN mRNA as a marker for de novo
fatty acid synthesis. We analyzed cell lines because breast tumor samples may contain adipocytes, which express high levels of LPL and FASN. We also sorted the breast cancer lines by their global gene expression signatures (21
). These signatures include the luminal type (estrogen receptor (ER) +), the basal, or “triple negative” type that lacks receptors for estrogen, progestin, and trastuzumab (22
), and the type with Her2/neu amplification. Only 6 breast cancer cell lines (HCC-2157, -1008, -1599, Du4475, SUM149, and SUM190) expressed high levels of LPL mRNA, and each of these exhibited the aggressive basal gene expression signature (Supplementary Fig. S1
). Expression of LPL mRNA by selected cell lines was verified by RT-PCR (), as was expression of CD36 mRNA (). LiSa-2 liposarcoma cells, which we previously demonstrated to exhibit the lipogenic phenotype (23
), also expressed LPL and CD36, as expected for a tumor cell derived from an adipocytic lineage. All of the cell types expressed substantial FASN mRNA (), and in the breast cancer cell lines this did not vary among the gene expression signatures (Supplementary Fig. S1
). Quantitative real time RT-PCR of representative lines confirmed that LiSa-2 liposarcoma and triple negative Du4475 breast cancer cells expressed the highest levels of LPL mRNA (). In contrast, prostate cancer cells, which are highly lipogenic (24
), expressed relatively low levels of LPL mRNA, and ER+ T47D and BT474 breast cancer cells expressed essentially none.
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 (more ...)
We examined conditioned tissue culture media for LPL enzyme activity, and it paralleled the levels of LPL mRNA (). LPL activity accumulated over time in culture media of LiSa-2 liposarcoma and Du4475 breast cancer cells (). In contrast, ER+ T47D, ER+ Her2/neu+ BT474 breast cancer cells, and fibroblasts did not secrete detectable lipase activity. Prostate cancer cells produced low levels of the enzyme. LPL activities in breast milk and murine striated muscle were substantially greater than those observed in any of the conditioned (72 h) media.
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 (more ...)
We found that available antibodies were not sufficiently specific to analyze LPL protein by immunohistochemistry. We therefore raised a mouse monoclonal antibody using a peptide representing residues 20–36 of the human enzyme as antigen. This antibody is highly specific (Supplemental Fig. S2
), and permitted detection of heparin sepharose-purified LPL from tissue culture media conditioned by Du4475 breast cancer and LiSa-2 liposarcoma cells ( upper). The band recognized by this antibody in western analysis of milk was verified to represent LPL by mass spectrometry. We could not detect LPL protein in media from ER+ breast or prostate cancer cells. Western analysis of a clinical breast tumor homogenate (50 mcg protein) without affinity purification revealed a single band exhibiting the same migration as that observed in milk ( lower).
It appeared possible that expression of heparanase could inactivate LPL, and thus could vitiate the metabolic relevance of LPL expression by tumors. We assessed expression of the heparanase gene (HPSE) using cDNA microarray data from 45 human breast cancer cell lines. This showed that the cells generally express very low levels of heparanase mRNA, as was the general case for LPL mRNA. We were intrigued to note that the subgroup of “triple negative” cell lines exhibiting substantial LPL expression also expressed the lowest levels of heparanase mRNA. Indeed, linear regression of the relationship between LPL and heparanase mRNAs in lines with the basal A signature revealed a statistically significant inverse correlation (p = 1.27 × 10−5,, R2 = 0.38). Thus, the coupling of high LPL with low heparanase expression appears to provide an advantage to the subset of cells that produce substantial LPL. Our examination of total and heparin-releasable LPL activity in a freshly-prepared breast tumor homogenate also reflects on this question, as heparin-releasable activity was readily detectable, arguing against depletion of a cell surface-bound LPL pool in breast tumors (see below).
We performed two experiments to determine whether cancer-associated LPL is bound to tumor cells by noncovalent interactions with cell surface heparan sulfate proteoglycans, using a protocol based on that of Cruz and coworkers (19
). First, we homogenized freshly-resected invasive breast cancer tissue shown to contain LPL immunoreactivity ( lower), and extracted equal aliquots with buffer containing heparin or not. LPL activity in the control sample was 1032 ± 8 without heparin, 768 ± 4 with heparin treatment (mean ± SE, nanomoles glycerol produced/g tumor/h, measured in triplicate; p < 0.0001). This represented a heparin-releasable fraction of 26% of the total tumor-associated LPL activity (represented by the portion of the bar labeled HR, left).
Second, we determined the heparin-releasable fraction of LPL in HeLa cells, and calculated turnover rates for cellular LPL pools ( right). Residual LPL activity in cell pellets was 13,260 ± 1,080 without, and 9,360 ± 820 with heparin exposure (units are nanomoles glycerol produced/flask/h, mean ± SEM, n= triplicate measurements/group; p < 0.04). We thus estimate that 29% of the HeLa cell-associated pool of LPL is heparin-releasable (indicated by HR on the graph), a fraction similar to that observed in the breast tumor sample. Measurement of LPL activity in culture media indicated that 36,000 ± 4,000 units of LPL activity were secreted/24 h. We therefore estimate that the total cellular LPL pool turns over 2.7 times/d, while the heparin-labile pool (3900 units/well) turns over, presumably by secretion, 9.2 times/d.
Our fetal calf serum (FCS) contained 660 mcg triglyceride/ml. LPL secreted by cells is removed when culture media are replaced, so the enzyme content in tissue culture never approaches that observed in tissues. We therefore assessed the functional significance of LPL by adding the enzyme to media containing 10% FCS and measuring cell accumulation. LPL activity under these culture conditions approximated that observed in mouse muscle. LPL enhanced the growth of T47D breast cancer cells, which do not express LPL, and of LiSa-2 liposarcoma cells, which do (). This effect of LPL was greatly reduced in media containing FCS that was nearly depleted of trigyceride (20 mcg/ml).
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 (more ...)
LNCaP prostate cancer cell growth was not accelerated by LPL addition. The ability of these cells to use exogenous triglyceride-derived fatty acids to maintain growth was revealed, however, in the presence of Soraphen A, a potent inhibitor of the lipogenic enzyme acetyl CoA-carboxylase (7
). The cells were rescued from Soraphen A-induced cytotoxicity by provision of LPL in the presence, but not in the absence, of lipoproteins (). Experiments using PC3 prostate cancer cells yielded similar results ().
In complementary studies we assessed the impact of siRNA-mediated knockdown of LPL mRNA on the growth of HeLa cells, which we previously reported to express the LPL gene (25
), and its interaction with inhibition of lipogenesis by Soraphen A. Two different siRNAs each caused > 90% disappearance of LPL mRNA, whereas a nonspecific siRNA was without effect (). Soraphen A caused a major inhibition of HeLa cell accumulation, and this effect was prevented by provision of LPL to the cultures (). Transfection of LPL siRNA A or B, but not of the nonspecific siRNA, significantly inhibited HeLa cell growth, and the anticancer effects of the two LPL siRNAs were further enhanced by exposure to Soraphen A.
We employed immunohistochemistry to assess the relevance of our findings in cultured cells to human tumors. We assessed the expression of markers of de novo
lipogenesis (FASN, THRSP (Spot 14, S14)), lipolysis (LPL), and exogenous FA uptake (CD36), in a panel of 147 breast, 24 liposarcoma, and 10 prostate tumor tissues (examples in ). FASN was cytosolic, in agreement with previous studies. S14, which promotes expression of the FASN gene (26
), was primarily nuclear, as reported (20
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 (more ...)
In contrast to our findings in breast cancer cell lines, LPL immunoreactivity was observed in all of the breast tumors examined, and, also in contrast to the cell lines, expression was not limited to triple negative tumors. Similarly, all liposarcoma and prostate tumors examined expressed readily detectable LPL by immunohistochemistry. Intracellular LPL demonstrated an asymmetric, perinuclear distribution suggestive of localization to the Golgi apparatus, as predicted for a glycosylated and secreted protein ( insets). As expected, extracellular LPL was found on the luminal surfaces of capillaries (Supplemental Fig. S2C
left). We stained tonsil tissue as a negative control, based on previous work showing undetectable LPL mRNA in lymphoid cells (25
). The lymphoid stroma indeed showed no staining except for scattered isolated monocytes, whereas the highly proliferative basal (stem cell) layer of the mucosal epithelium overlying the tonsil unexpectedly showed a strong signal (Supplemental Fig. S2C
The majority of tumors also stained for CD36 (). Interestingly, two distinct staining patterns were observed in breast cancer tissue. Of 144 evaluable cores, 42 exhibited diffuse cytoplasmic staining without accentuation at the plasma membrane (, left upper), whereas 100 also demonstrated a strong cell surface signal (lower panel). Only 2 breast cancer cases were devoid of staining. Chi square analysis demonstrated a statistically significant difference in the prevalence of the membranous staining pattern between the “triple negative” and ER+ breast cancers (42 vs. 76%, p < 0.02).
Of 25 liposarcoma cases, 21 stained for CD36, almost all in a mixed cytoplasmic plus plasma membrane pattern (, middle), including all 9 cases of well-differentiated liposarcoma. Of 9 evaluable prostate cancers, 4 showed focally positive staining in a mixed cytoplasmic and plasma membrane pattern (, right), while 5 cases scored negative for CD36.
Expression of LPL by breast cancer cells suggested the possibility that the cells could use the enzyme not only to hydrolyze extracellular triglyceride, but also for receptor-mediated endocytosis of triglyceride-rich lipoproteins. This process employs LPL as a bridge between the cell surface receptor syndecan-1 and the lipoprotein (28
). RT-PCR revealed readily detectable syndecan-1 mRNA from DU4475 breast cancer cells, while LiSa-2 and T47D exhibited a faint signal (Supplementary Fig. S3A
). We incubated fibroblasts and DU4475 cells with fluorescently-labeled VLDL particles, and assessed for cellular uptake using confocal microscopy. Abundant uptake was observed in fibroblasts (Fig. S3B
), but not in DU4475 cells (Fig. S3C
). Occasional fluorescence was detected on the cell surface (Fig. S3D
), but never within the breast cancer cells.