Cholesterol is needed for the biogenesis and maintenance of fluidity of cell membranes (41
). It is also a central component of lipid rafts, specialized microdomains of the plasma membrane that serve as organizing centers for the assembly of signaling molecules (45
). Therefore, rapidly proliferating cancer cells with highly activated signal transduction networks, such as GBM cells, are likely to have an enhanced requirement for cholesterol (48
). However, the molecular mechanisms by which GBM cells obtain sufficient cholesterol and the potential therapeutic targetability of this process are not well understood. Here, through integrated analyses in GBM cell lines, xenograft models and GBM clinical samples, including from patients treated with the EGFR tyrosine kinase inhibitor lapatinib, we have uncovered an EGFRvIII-activated, PI3K/SREBP-1-dependent tumor survival pathway involving LDLR. The present studies begin to shed light on the molecular mechanism by which an oncogene and its signal transduction effectors alter the metabolic circuitry to meet the enhanced tumor cell demand for cholesterol.
Most attempts to target cholesterol metabolism in cancer have focused on the use of the statin class of HMG-CoA reductase inhibitors that block the rate limiting step in de novo cholesterol synthesis (51
). In non-cancerous cells, the transcription factors SREBP and LXR maintain cholesterol homeostasis through complementary pathways of feedback inhibition and feed-forward activation. Thus, LDLR expression is suppressed by high cellular cholesterol levels through both inactivation of SREBPs and activation of the LXR-IDOL axis (27
). We have shown here that GBM cells have devised a mechanism to subvert the normal pathways for feedback inhibition via the EGFRvIII and PI3K-dependent activation of SREBP-1. Twenty years ago, Rudling and colleagues detected elevated LDL binding and LDLR expression in GBM relative to normal brain (53
). However, the molecular basis for elevated LDLR expression, and its potential therapeutic implications, including the potential effect of sensitivity to statins, has not been tested. Here, we show that constitutive EGFRvIII/PI3K-signaling through SREBP-1 results in unrestrained LDLR expression (), thus potentially rendering tumor cells resistant to HMG-CoA reductase inhibitors(14
). Consistent with this model, in the absence of extracellular cholesterol, atorvastatin significantly inhibited the growth and promoted cell death of GBM cells (Supplemental Figure 6
). These findings provide an explanation for why many tumor cells are resistant to statin treatment, and suggest alternative routes towards targeting cholesterol homeostasis in cancer.
In addition to cholesterol, LDL also contains Apo B-100, fatty acids and phospholipids (54
), raising the possibility that factors in addition to cholesterol, may be required by GBMs for optimal growth. Although we cannot formally exclude this possibility, we observed that overexpression of IDOL, which decreases LDLR expression (), and in combination with atorvastatin treatment, which inhibits endogenous cholesterol synthesis, show remarkable anti-tumor synergy, although neither agent is effective alone (Supplemental Figure 7
). These data suggest that cholesterol is the critical ingredient of LDL required by GBM cells, and that enhanced ability to take up exogenous cholesterol though LDLR renders statins ineffective.
PI3K signaling is hyperactivated as a consequence of RTK amplifications and activating mutations, PTEN loss, PI3K point mutations and other genetic lesions, providing a core oncogenic pathway in many cancers, including up to 90% of GBMs (6
). EGFR amplification, and EGFRvIII activating mutation are the most common oncogenes promoting PI3K signaling in GBM (1
). However, other RTKs that can be co-expressed in GBM, including some that may be upregulated after EGFR inhibitor therapies, like c-MET, PDGFR-alpha and PDGFR-beta, can also engage PI3K signaling, resulting in EGFR inhibitor resistance (12
). This prompted us to determine whether other PI3K-activating RTKs also promote LDLR expression. Consistent with this model, we detected a strong correlation between c-MET and PDGFR-beta expression and SREBP-1 and LDLR (Supplemental Figure 3, A–D
). More importantly, addition of HGF can potently stimulate SREBP-1 cleavage and LDLR expression in c-MET-expressing GBM cells (Supplemental Figure 3E
), suggesting that other PI3K-activating lesions can also promote LDLR expression. These results broaden the potential spectrum of tumors that may be susceptible to anti-LDLR-mediated therapies, including LXR agonists. Furthermore, the PI3K pathway is hyperactivated not only in GBM, but also in many other cancers including breast, ovarian, endometrial, lung, prostate, renal and lymphocyte (5
). Therefore, we hypothesize that the mechanisms discovered here in GBM may be relevant to many PI3K-driven cancers. Future studies will be needed to determine whether PI3K hyperactivation promotes enhanced LDLR expression and dependence on LDL in other cancers, and whether this is a targetable mechanism across multiple cancer types.
mTORC1 appears to be critical for linking PI3K signaling with tumor metabolism (16
). SREBP-1 expression and/or activity are regulated by PI3K/Akt signaling through mTORC1 in hepatocytes (61
), mouse embryonic fibroblasts (62
) and in Drosphila (63
). Further, mTORC1 activation of SREBP-1 has been shown to be essential for regulating lipid and sterol biogenesis (62
). However, these studies have been conducted largely in non-cancerous cells; the role of mTORC1 in regulating SREBP-1 and cellular metabolism in cancer remains to be elucidated.
Surprisingly, we have found that SREBP-1 activation is rapamycin insensitive, calling into question its regulation by mTOR in GBM. In pre-clinical models () and in GBM patients treated with rapamycin (14
), we have shown that SREBP-1 activation, and consequent LDLR expression, are rapamycin resistant (). There are two potential explanations for these results. PI3K signaling to SREBP-1 may not require mTOR, perhaps due to an alteration in the molecular circuitry linking Akt with SREBP-1 in cancer cells. Alternatively, SREBP-1 activity may be mTOR-dependent, but rapamycin-insensitve due to incomplete inhibition of either mTORC1 or mTORC2 signaling. Further studies are needed to determine whether SREBP-1 is regulated by mTOR in cancer, to dissect its metabolic consequences, and to determine whether mTOR kinase inhibitors can block PI3K/Akt mediated lipogenesis through SREBP-1.
The nuclear receptor LXR emerges from these studies as a potential adjuvant drug target in GBM. Although we have previously shown that forced activation of the LXR pathway with highly efficacious synthetic agonists inhibits the growth of rapidly dividing primary cells, the relevance of this effect for transformed cells has not been investigated. Here we show that the synthetic LXR agonist GW3965 potently suppresses GBM growth and induces apoptosis in a mouse model (), and we demonstrate enhanced efficacy in EGFRvIII-expressing GBM cells (). Interestingly, we find that IDOL-mediated degradation of LDLR is necessary, but not sufficient, to induce GBM cell apoptosis (). Because cellular cholesterol levels depend on the integrated activities of the uptake, efflux and synthesis pathways (44
), LXR agonists may be highly beneficial because of their ability to coordinately target two of the three aspects of cholesterol regulation (27
). Such drugs not only block exogenous LDL uptake, they also actively promote cholesterol removal from cells and intracellular distribution out of the endoplasmic reticulum (ER) (28
Pharmacokinetic and toxicity studies have demonstrated that GW3965 may induce elevated hepatic triglycerides (25
). Therefore, new synthetic LXR agonists are being developed that similarly activate LXR without producing the same degree of hepatic triglyceride induction. The fatty acid synthase inhibitor C75 promoted an additive anti-tumor growth effect when administered with GW3965, suggesting a potential role for combination therapy (Supplemental Figure 8
). Future studies will be needed to assess the efficacy and clinical utility of those compounds as potential clinical candidates as they become available for testing.
In summary, our integrated studies in GBM cell lines, mouse models and human clinical trial samples have delineated an EGFRvIII-activated, PI3K/SREBP-1-dependent tumor survival pathway through LDLR (). Our data also suggest that LXR-IDOL-LDLR axis is a common targetable pathway in multiple tumor types (; Supplemental Figure 5
). Consistent with this hypothesis, activation of LXR in different types of cancer cell lines resulted in significant cell death (Supplemental Figure 9
), raising the possibility that this axis may be a compelling drug target in multiple cancers. Further delineation of the molecular mechanisms by which PI3K signaling differentially regulates tumor cell metabolism will inform a better understanding of the links between genetic alterations and cellular metabolism in cancer, and may potentially lead to more effective, less toxic treatments.