An LXR Agonist Promotes Glioblastoma Cell Death through Inhibition of an EGFR/AKT/SREBP-1/LDLR–Dependent Pathway

In the paper

“Cancer cells preferentially metabolize glucose by aerobic glycolysis, a phenomenon known as the Warburg effect.”

What the sources say

• ?Not checked — no full text of the sourcecites [16] DeBerardinis, 2008, The biology of cancer: metabolic reprogramming fuels cell growth and proliferation, Cell Metab source
• Supported

  In “Proliferating Mammalian Cells Exhibit Anabolic Metabolism,” the paper says most cancer cells produce large amounts of lactate regardless of oxygen availability and calls this metabolism “aerobic glycolysis.” It also identifies this pattern as Warburg’s observation. The claim is a fair paraphrase, though the paper specifies “most” rather than all cancer cells. (LLM, conf=0.93)

  cites [17] Heiden, 2009, Understanding the Warburg effect: the metabolic requirements of cell proliferation, Science source
• ?Not checked — no full text of the sourcecites [18] Kroemer, 2008, Tumor cell metabolism: cancer's Achilles' heel, Cancer Cell source
• ?Not checked — no full text of the sourcecites [19] Menendez, 2007, Fatty acid synthase and the lipogenic phenotype in cancer pathogenesis, Nat Rev Cancer source

In the paper

“PI3K signaling is hyperactivated in nearly 90% of GBMs, commonly as a consequence of EGFR amplification and activating mutation (EGFRvIII), and loss of the PTEN tumor suppressor protein, a negative regulator of PI3K signaling (6–8).”

What the sources say

• Partly supported

  The Results section reports that 86% of GBM samples had a genetic event in the RTK/PI3K pathway, and documents EGFR amplification, EGFRvIII, and PTEN deletions or mutations (page not provided). That supports the approximate prevalence and the named genetic alterations, but the paper does not establish that PI3K signaling was measured as hyperactivated in nearly 90% of tumors or that PTEN protein was lost in those cases. (LLM, conf=0.88)

  cites [6] Cancer Genome Atlas Research Network, 2008, Comprehensive genomic characterization defines human glioblastoma genes and core pathways, Nature sourceExperimental — accuracy not yet measured
• Partly supported

  The Integration of sequencing, copy number, and expression analyses section reports alterations in PI3K/PTEN-pathway genes in 50% of tumors and identifies EGFR and PTEN alterations in 30–40% of GBMs. It does not report PI3K signaling hyperactivation in nearly 90% of GBMs or establish EGFRvIII and PTEN protein loss as its common causes. Page numbers are unavailable in our copy. (LLM, conf=0.87)

  cites [7] Parsons, 2008, An integrated genomic analysis of human glioblastoma multiforme, Science sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [8] Chakravarti, 2004, The prognostic significance of phosphatidylinositol 3-kinase pathway activation in human gliomas, J Clin Oncol source

In the paper

“LXRs respond to excess cellular cholesterol by promoting ABCA1- and ABCG1-dependent cholesterol efflux and by inhibiting LDLR protein expression through induction of the E3 ubiquitin ligase IDOL (27).”

What the sources say

• Supported

  The paper states that LXRs respond to cellular cholesterol excess and that LXR targets ABCA1 and ABCG1 promote cholesterol efflux. It also reports that LXR induces the E3 ubiquitin ligase Idol, which targets LDLR for degradation and reduces LDLR protein levels (passages labeled Abstract; no page numbers provided). (LLM, conf=0.94)

  cites [27] Zelcer, 2009, LXR regulates cholesterol uptake through Idol-dependent ubiquitination of the LDL receptor, Science source

In the paper

“Despite the compelling nature of EGFR as a drug target in GBM, the EGFR inhibitors gefitinib and erlotinib have failed to demonstrate efficacy (9–11), in large part as a consequence of persistent PI3K signaling due to PTEN loss and/or coactivation of other receptor tyrosine kinases (9, 10, 12).”

What the sources say

• Partly supported

  The Abstract (page unavailable) reports that only 10–20% of patients respond to EGFR kinase inhibitors, but it also says a small subgroup benefits from gefitinib or erlotinib. The in vitro studies section (page unavailable) links PTEN loss to resistance, while noting both Akt-dependent and Akt-independent mechanisms. Our copy does not establish coactivation of other receptor tyrosine kinases as a cause of resistance, or show that persistent PI3K signaling largely explains the drugs’ limited efficacy. (LLM, conf=0.87)

  cites [9] Mellinghoff, 2005, Molecular determinants of the response of glioblastomas to EGFR kinase inhibitors, N Engl J Med sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [10] Haas-Kogan, 2005, Epidermal growth factor receptor, protein kinase B/Akt, and glioma response to erlotinib, J Natl Cancer Inst source
• Partly supported

  In “Measurement of Therapeutic Effect, Time to Progression, and Survival” (page unavailable), the gefitinib trial reports no complete or partial responses and frequent progression, supporting limited demonstrated efficacy in recurrent GBM. But “Factors Related to Response, Progression, and Survival” (page unavailable) says erlotinib seemed to show activity; it does not establish persistent PI3K signaling, PTEN loss, or coactivation of other receptor tyrosine kinases as the reason for limited efficacy. (LLM, conf=0.85)

  cites [11] Rich, 2004, Phase II trial of gefitinib in recurrent glioblastoma, J Clin Oncol sourceExperimental — accuracy not yet measured
• Partly supported

  On p. 1, the paper reports that PTEN loss can make EGFR inhibitors ineffective by sustaining PI3K activity and proposes that inputs from other RTKs may also limit their efficacy. Its cell-line experiments on p. 2 show that combined RTK inhibition suppresses signaling where single-agent inhibition does not. The paper does not establish that these mechanisms are largely responsible for gefitinib and erlotinib's lack of clinical efficacy in GBM; it presents the broader clinical explanation as a possibility. (LLM, conf=0.83)

  cites [12] Stommel, 2007, Coactivation of receptor tyrosine kinases affects the response of tumor cells to targeted therapies, Science sourceExperimental — accuracy not yet measured

In the paper

“Cholesterol metabolism in mammals is controlled through the coordinated action of SREBP and LXR transcription factors (24–26).”

What the sources say

• ?Not checked — no full text of the sourcecites [24] Brown, 1997, The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor, Cell source
• Partly supported

  The section “LXRs are master regulators of whole-body cholesterol homeostasis” supports LXR’s role in cholesterol metabolism. “LXR signaling in intermediary metabolism and energy balance” says LXR upregulates SREBP-1c, but describes that relationship as regulating hepatic lipogenesis, not coordinated control of cholesterol metabolism by both factors. (LLM, conf=0.84)

  cites [25] Zelcer, 2006, Liver X receptors as integrators of metabolic and inflammatory signaling, J Clin Invest sourceExperimental — accuracy not yet measured
• Partly supported

  The Introduction identifies LXRs as central regulators of cholesterol metabolism in mammals. It also links SREBP and LXR transcriptional responses to sterol availability during lymphocyte activation, but does not establish their coordinated control of mammalian cholesterol metabolism generally. (LLM, conf=0.84)

  cites [26] Hong, 2008, Coordination of inflammation and metabolism by PPAR and LXR nuclear receptors, Curr Opin Genet Dev sourceExperimental — accuracy not yet measured

In the paper

“We recently showed that mutant EGFRvIII expressing GBMs promote lipogenesis through PI3K-dependent activation of the master transcriptional regulator SREBP-1, and we demonstrated that this signal was required for tumor survival in vivo (14).”

What the sources say

• Partly supported

  The Results sections on EGFR–PI3K–Akt signaling and SREBP-1 knockdown show that EGFRvIII promotes SREBP-1 activation and fatty-acid synthesis, and that SREBP-1 knockdown kills EGFRvIII-bearing cells in culture. The in vivo Results show that the lipogenesis inhibitor C75 promotes apoptosis in EGFRvIII-bearing tumors, but do not directly show that PI3K-dependent SREBP-1 activation itself is required for tumor survival in vivo. Page numbers are unavailable in our copy. (LLM, conf=0.84)

  cites [14] Guo, 2009, EGFR signaling through an Akt-SREBP-1-dependent, rapamycin-resistant pathway sensitizes glioblastomas to antilipogenic therapy, Sci Signal sourceExperimental — accuracy not yet measured

In the paper

“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, 56–58).”

What the sources say

• Partly supported

  The Introduction (page unavailable) describes PI3K-pathway activation across many cancers and identifies pathway-related alterations in breast, prostate and brain cancers; it also discusses endometrial and lung cancers. Our copy does not substantiate the full list: the searched passages do not identify ovarian, renal or lymphocyte cancers as examples of PI3K-pathway hyperactivation. (LLM, conf=0.85)

  cites [5] Wong, 2010, Targeting the PI3K signaling pathway in cancer, Curr Opin Genet Dev sourceExperimental — accuracy not yet measured
• Partly supported

  The Introduction describes widespread PI3K pathway activation in cancer and identifies breast, prostate and brain cancers; the section on concurrent alterations also identifies endometrial cancer, and the paper discusses PI3K-driven lung cancer. The passages do not establish the claim for ovarian, renal or lymphocyte cancers specifically. Page numbers are not available in our copy. (LLM, conf=0.86)

  cites [56] Wong, Targeting the PI3K signaling pathway in cancer, Curr Opin Genet Dev sourceExperimental — accuracy not yet measured
• Partly supported

  The Introduction and Known mutations in the pathway sections describe widespread PI3K-pathway dysregulation in cancer. The glioblastoma, HER2, and PTEN sections provide examples involving GBM and breast, ovarian, endometrial, lung, and prostate cancers. Our copy does not establish the claim for renal cancer or lymphocyte-derived cancer; its Immune cells section discusses PI3K activation in immune cells within the tumor environment, not cancers of those cells. No page numbers were available in the indexed passages. (LLM, conf=0.88)

  cites [57] Yuan, 2008, PI3K pathway alterations in cancer: variations on a theme, Oncogene sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [58] Luo, 2003, Targeting the PI3K-Akt pathway in human cancer: rationale and promise, Cancer Cell source

In the paper

“EGFR amplification, and EGFRvIII activating mutation are the most common oncogenes promoting PI3K signaling in GBM (1, 6).”

What the sources say

• Supported

  The EGFR section (page unavailable) reports EGFR amplification in approximately 40% of GBMs and EGFRvIII in 20–30%, calling EGFRvIII the most common EGFR mutant. The Mitogenic signaling pathways section (page unavailable) explains that PI3Ks are recruited to activated receptor tyrosine kinases, and the paper associates PI3K activation profiles with EGFRvIII expression. Together, these passages support the claim’s characterization of these as common EGFR alterations promoting PI3K signaling in GBM. (LLM, conf=0.88)

  cites [1] Furnari, 2007, Malignant astrocytic glioma: genetics, biology, and paths to treatment, Genes Dev source
• Partly supported

  The Results section on EGFR family activation (page unavailable) reports frequent EGFR activation in primary GBM, focal EGFR amplification, and EGFR alterations in 41 of 91 sequenced cases; it calls vIII the most commonly described EGFR event. The core-pathways Results section places EGFR in the RTK/PI3K pathway, but the paper does not establish that EGFR amplification and EGFRvIII are together the most common oncogenic drivers of PI3K signaling, or give a separate frequency for vIII. (LLM, conf=0.83)

  cites [6] Cancer Genome Atlas Research Network, 2008, Comprehensive genomic characterization defines human glioblastoma genes and core pathways, Nature sourceExperimental — accuracy not yet measured

In the paper

“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, 8, 55).”

What the sources say

• Partly supported

  The Results (Integrative analyses define core pathways required for GBM pathogenesis; page unavailable) report that 86% of sampled GBMs had a genetic event in the combined RTK/PI3K pathway, including RTK aberrations and PTEN lesions. The Discussion also describes RTK/RAS/PI3K deregulation as common in GBM. Those passages support a core pathway and an approximate ‘up to 90%’ figure for pathway alterations, but they do not establish that PI3K signaling itself was hyperactivated in that proportion of GBMs or establish the broader prevalence claim across many cancers. (LLM, conf=0.86)

  cites [6] Cancer Genome Atlas Research Network, 2008, Comprehensive genomic characterization defines human glioblastoma genes and core pathways, Nature sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [8] Chakravarti, 2004, The prognostic significance of phosphatidylinositol 3-kinase pathway activation in human gliomas, J Clin Oncol source
• ?Not checked — no full text of the sourcecites [55] Choe, 2003, Analysis of the phosphatidylinositol 3′-kinase signaling pathway in glioblastoma patients in vivo, Cancer Res

In the paper

“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).”

What the sources say

• Partly supported

  The Discussion (page not provided) links LXR-induced ABCG1 expression to sterol efflux and describes redistribution of sterols out of the ER, though it presents the effect of LXR agonists on these processes as a likely mechanism rather than a directly measured outcome. The Results section on cholesterol homeostasis (page not provided) says added LDL did not change proliferation; it does not show that LXR agonists block LDL uptake. (LLM, conf=0.86)

  cites [28] Bensinger, 2008, LXR signaling couples sterol metabolism to proliferation in the acquired immune response, Cell sourceExperimental — accuracy not yet measured

In the paper

“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).”

What the sources say

• Supported

  The Results (Fig. 1B; page not provided) show that insulin-induced SREBP-1c mRNA expression in primary rat hepatocytes was blocked by inhibitors of PI3K, Akt, and mTORC1. Citation (61) is attached to the hepatocyte portion of the sentence, not the claims about mouse embryonic fibroblasts or Drosophila. (LLM, conf=0.93)

  cites [61] Li, 2010, Bifurcation of insulin signaling pathway in rat liver: mTORC1 required for stimulation of lipogenesis, but not inhibition of gluconeogenesis, Proc Natl Acad Sci U S A source
• Partly supported

  Citation (62) is attached to the mouse embryonic fibroblast portion of the sentence. In the Results section on SREBP, the paper reports rapamycin-sensitive increases in processed SREBP1 and Srebp1 transcripts in Tsc2-deficient MEFs, supporting regulation through mTORC1. It does not establish the PI3K/Akt step in those MEFs; it cites a separate study for the observation that activated Akt increases processed SREBP1. (LLM, conf=0.83)

  cites [62] Duvel, 2010, Activation of a metabolic gene regulatory network downstream of mTOR complex 1, Mol Cell sourceExperimental — accuracy not yet measured
• Partly supported

  For the Drosophila portion of the citing sentence, the Results section (“SREBP Is a Target of the PI3K Pathway in Drosophila”) reports that PI3K/Akt signaling activates dSREBP function. But the Discussion says it remains to be investigated whether dSREBP regulation downstream of PI3K requires TOR, so the claimed mTORC1 link in flies is not established by this paper. (LLM, conf=0.90)

  cites [63] Porstmann, 2008, SREBP activity is regulated by mTORC1 and contributes to Akt-dependent cell growth, Cell Metab sourceExperimental — accuracy not yet measured

In the paper

“Pharmacokinetic and toxicity studies have demonstrated that GW3965 may induce elevated hepatic triglycerides (25).”

What the sources say

• Partly supported

  The section “LXR signaling in intermediary metabolism and energy balance” (page unavailable) reports that synthetic LXR agonists elevate liver triglycerides in mice. It does not identify GW3965 or describe pharmacokinetic and toxicity studies, so it supports the general risk but not the compound-specific attribution. (LLM, conf=0.85)

  cites [25] Zelcer, 2006, Liver X receptors as integrators of metabolic and inflammatory signaling, J Clin Invest sourceExperimental — accuracy not yet measured

In the paper

“Further, mTORC1 activation of SREBP-1 has been shown to be essential for regulating lipid and sterol biogenesis (62).”

What the sources say

• Supported

  The Results section reports that mTORC1 increases active SREBP1 and that Srebp1 knockdown reduces mTORC1-dependent lipid biosynthesis in Tsc2−/− cells. It also reports that SREBP1 and SREBP2 are required for mTORC1-induced expression of fatty-acid and sterol biosynthesis genes, while noting that SREBP2 has the more dominant role for sterol genes. (LLM, conf=0.91)

  cites [62] Duvel, 2010, Activation of a metabolic gene regulatory network downstream of mTOR complex 1, Mol Cell source

In the paper

“These cell lines have previously been reported (9, 29).”

What the sources say

• Partly supported

  The U87MG Model section (page unavailable) reports U87MG cells engineered to express EGFR or EGFRvIII, including PTEN-overexpressing cells. Other Cell-Line Models (page unavailable) reports A431 cells. Our copy does not report LN229, T98, H1975, or HeLa, so this source supports only part of the listed cell lines. (LLM, conf=0.88)

  cites [9] Mellinghoff, 2005, Molecular determinants of the response of glioblastomas to EGFR kinase inhibitors, N Engl J Med sourceExperimental — accuracy not yet measured
• Partly supported

  The Results and Discussion section identifies U87MG and the U87-EGFR, U87-EGFRvIII, and U87-PTEN-EGFR cell lines, supporting that portion of the identification citation. Our copy does not identify A431, LN229, T98, H1975, or HeLa, so it does not support the full list. Page numbers were not provided in the indexed passages. (LLM, conf=0.88)

  cites [29] Wang, 2006, Mammalian target of rapamycin inhibition promotes response to epidermal growth factor receptor kinase inhibitors in PTEN-deficient and PTEN-intact glioblastoma cells, Cancer Res sourceExperimental — accuracy not yet measured

In the paper

“Because cellular cholesterol levels depend on the integrated activities of the uptake, efflux and synthesis pathways (44)”

What the sources say

• Supported

  The “Transport of cholesterol into and out of cells” section describes cholesterol uptake, cellular synthesis, and export. The “Cellular cholesterol homeostasis” section describes regulation of synthesis in response to cholesterol levels. Together, these passages support the paraphrase; page numbers were not provided in our copy. (LLM, conf=0.90)

  cites [44] Maxfield, 2010, Cholesterol, the central lipid of mammalian cells, Curr Opin Cell Biol source

In the paper

“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).”

In the paper

“Sixty percent growth inhibition was detected in EGFRvIII expressing GBM cells; only half as much was seen in parental U87 cells (Fig. 3A).”

In the paper

“As shown in Figure 2E, we detected decreased LDLR expression after lapatinib treatment, in association with decreased p-EGFR, p-Akt and nuclear SREBP-1 staining (Figure 2E).”

In the paper

“Indeed, treatment of U87 and U87-EGFRvIII GBM cells for 4 days with the LXR agonist GW3965 resulted in dose-dependent inhibition of growth and promotion of tumor cell death.”

In the paper

“overexpression of IDOL, which decreases LDLR expression (Figure, 7D), 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).”

In the paper

“We detected LDLR expression in some tumors that did not stain positive for p-EGFR. However, these samples showed evidence for PI3K pathway activation, as determined by p-Akt staining (p<0.001) (Figure 2C).”

In the paper

“In U251 GBM cells, a cell line that expresses relatively little EGFR but expresses abundant levels of c-Met, HGF stimulated Met phosphorylation and promoted SREBP-1 cleavage and LDLR expression (Supplemental Figure. 3E).”

In the paper

“Erlotinib treatment inhibited EGFR and Akt phosphorylation, and induced potent suppression of LDLR expression (Figure 1B).”

In the paper

“In contrast, the allosteric mTOR inhibitor rapamycin did not suppress EGF-mediated LDLR expression (Figure 1F), nor did it suppress SREBP-1 cleavage.”

In the paper

“Erlotinib (10 uM), LY294002 (10 uM) and Akti 1/2 (5 uM) treatment, targeted to block EGFR, PI3K and Akt signaling respectively, each produced potent suppression of EGF-mediated SREBP-1 cleavage and LDLR expression (Figure 1F).”

In the paper

“GW3965-mediated LDLR degradation in GBM cells took longer, and required a higher drug dose, than did ABCA1 induction (Figure 5, C and D).”

In the paper

“GW3965 treatment strongly induced ABCA1 expression and reduced LDLR expression (Figure 7A).”

In the paper

“GW3965 treatment promoted dose-dependent increases in ABCA1 and IDOL, with a concomitant decrease in LDLR protein level (Figure 5, A–D; Supplemental Figure 5).”

In the paper

“Neither LDLR knockdown alone, nor IDOL overexpression alone, were sufficient to promote GBM cell death (Figure 6).”

In the paper

“Low dose GW3965 (1 or 2 uM) induced ABCA1, but did not diminish LDLR expression or cause GBM cell death (Figure 6, A–C).”

In the paper

“Immunoblot analysis of EGFRvIII-expressing GBMs showed abundant LDLR expression, but expression was scarcely detectable in tumors arising from parental U87MG cells (Figure 1A).”

In the paper

“Increased LDLR mRNA was detectable within 30 minutes of EGF stimulation (Supplementary Figure 1A), resulting in increased LDLR protein expression detectable two hours after stimulation (Figure 1E).”

In the paper

“Here we show that the synthetic LXR agonist GW3965 potently suppresses GBM growth and induces apoptosis in a mouse model (Figure 7)”

In the paper

“we find that IDOL-mediated degradation of LDLR is necessary, but not sufficient, to induce GBM cell apoptosis (Figure 6).”

In the paper

“SREBPs promote the expression of genes involved in cholesterol synthesis and enhance the uptake of extracellular cholesterol by inducing expression of the LDL receptor (LDLR).”

In the paper

“These data demonstrate that an LXR agonist potently inhibits GBM growth and promotes tumor cell death in vivo.”

In the paper

“Taken together, these results demonstrate that EGFR signaling through Akt is associated with nuclear SREBP-1 and LDLR expression in GBM patients, and other PI3K-activating RTKs can also potentially promote LDLR expression.”

In the paper

“Twenty years ago, Rudling and colleagues detected elevated LDL binding and LDLR expression in GBM relative to normal brain (53).”

What the sources say

• ?Not checked — no full text of the sourcecites [53] Rudling, 1990, Low density lipoprotein receptor activity in human intracranial tumors and its relation to the cholesterol requirement, Cancer Res

In the paper

“mTORC1 appears to be critical for linking PI3K signaling with tumor metabolism (16, 59, 60).”

What the sources say

• ?Not checked — no full text of the sourcecites [16] DeBerardinis, 2008, The biology of cancer: metabolic reprogramming fuels cell growth and proliferation, Cell Metab source
• ?Not checked — no full text of the sourcecites [59] Engelman, 2006, The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism, Nat Rev Genet source
• ?Not checked — no full text of the sourcecites [60] Yecies, 2011, mTOR links oncogenic signaling to tumor cell metabolism, J Mol Med source

In the paper

“Tumor specimens were obtained according to a protocol approved by the Institutional Review Board of UCLA.”

In the paper

“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.”

In the paper

“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.”

In the paper

“SCID/Beige mice were bred and kept under defined-flora pathogen-free conditions at the AALAC-approved Animal Facility of the Division of Experimental Radiation Oncology, UCLA.”