Low-density Lipoprotein Receptor Knockdown Research Articles

PAQR3 facilitates the ferroptosis of diffuse large B-cell lymphoma via the regulation of LDLR-mediated PI3K/AKT pathway.

Progesterone and adiponectin receptor 3 (PAQR3) has been found to regulate tumor progression by mediating cell ferroptosis. However, whether PAQR3 mediates ferroptosis in diffuse large B-cell lymphoma (DLBCL) needs further investigation. The mRNA and protein levels of PAQR3 and low-density lipoprotein receptor (LDLR) were assessed by qRT-PCR and WB assays. Cell proliferation was detected by MTT assay and EdU assay. Shrunken mitochondria was counted under transmission electron microscope. Cell ferroptosis was evaluated by measuring the levels of malondialdehyde, reactive oxygen species, glutathione, Fe2+ , and the protein expression of ferroptosis-related markers. PAQR3 and LDLR interaction was confirmed by RIP assay and pull-down assay. Our study showed that PAQR3 was underexpressed, while LDLR was overexpressed in DLBCL tissues and cells. Functionally, PAQR3 overexpression or LDLR knockdown restrained DLBCL cell proliferation and enhanced ferroptosis. Mechanistically, PAQR3 reduced LDLR expression by inhibiting its mRNA stability. Meanwhile, LDLR overexpression reversed PAQR3-mediated the promoting on DLBCL cell ferroptosis, and LY294002 (PI3K/AKT inhibitor) eliminated the inhibiting effects of LDLR overexpression on DLBCL cell ferroptosis. Additionally, excessive PAQR3 reduced DLBCL tumor growth by enhancing tumor cell ferroptosis through LDLR-mediated PI3K/AKT pathway. In conclusion, our data suggested that PAQR3 restrained DLBCL progression by aggravating ferroptosis, which was achieved by inhibiting LDLR expression to repress PI3K/AKT pathway.

Anlotinib affects systemic lipid metabolism and induces lipid accumulation in human lung cancer cells

BackgroundAnlotinib has demonstrated encouraging clinical outcomes in the treatment of lung cancer, soft tissue sarcoma and thyroid carcinoma. Several clinical studies have shown a relationship between anlotinib treatment and the occurrence of hyperlipidemia. The fundamental mechanisms, however, are still largely unclear. Here, the effect of anlotinib on lipid metabolism in an animal model and human cancer cells was evaluated and the role of lipid metabolism in the antitumor efficacy of anlotinib was investigated.MethodsThe C57BL/6 J mouse model as well as A549 and H460 human lung cancer cell lines were used to examine the impact of anlotinib on lipid metabolism both in vivo and in vitro. Levels of triglycerides, high-density lipoprotein, low-density lipoprotein (LDL), and total cholesterol in serum or cell samples were determined using assay kits. The expression levels of crucial genes and proteins involved in lipid metabolism were measured by quantitative RT-PCR and Western blotting. Furthermore, exogenous LDL and knockdown of low-density lipoprotein receptor (LDLR) were used in H460 cells to investigate the relevance of lipid metabolism in the anticancer efficacy of anlotinib.ResultsAnlotinib caused hyperlipidemia in C57BL/6 J mice, possibly by downregulating hepatic LDLR-mediated uptake of LDL cholesterol. AMP-activated protein kinase and mammalian target of rapamycin inhibition may also be involved. Additionally, anlotinib enhanced sterol response element binding protein 1/2 nuclear accumulation as well as upregulated LDLR expression in A549 and H460 cells, which may be attributable to intracellular lipid accumulation. Knockdown of LDLR reduced intracellular cholesterol content, but interestingly, anlotinib significantly improved intracellular cholesterol accumulation in LDLR-knockdown cells. Both exogenous LDL and LDLR knockdown decreased the sensitivity of cells to anlotinib.ConclusionsAnlotinib modulates host lipid metabolism through multiple pathways. Anlotinib also exerts a significant impact on lipid metabolism in cancer cells by regulating key transcription factors and metabolic enzymes. In addition, these findings suggest lipid metabolism is implicated in anlotinib sensitivity.

Low-Density Lipoprotein Receptor Is a Key Driver of Aggressiveness in Thyroid Tumor Cells.

We previously described the role of low-density lipoprotein (LDL) in aggressiveness in papillary thyroid cancer (PTC). Moreover, the MAPK signaling pathway in the presence of BRAF V600E mutation is associated with more aggressive PTC. Although the link between MAPK cascade and LDL receptor (LDLR) expression has been previously described, it is unknown whether LDL can potentiate the adverse effects of PTC through it. We aimed to investigate whether the presence of LDL might accelerate the oncogenic processes through MAPK pathway in presence or absence of BRAF V600E in two thyroid cell lines: TPC1 and BCPAP (wild-type and BRAF V600E, respectively). LDLR, PI3K-AKT and RAS/RAF/MAPK (MEK)/ERK were analyzed via Western blot; cell proliferation was measured via MTT assay, cell migration was studied through wound-healing assay and LDL uptake was analyzed by fluorometric and confocal analysis. TPC1 demonstrated a time-specific downregulation of the LDLR, while BCPAP resulted in a receptor deregulation after LDL exposition. LDL uptake was increased in BCPAP over-time, as well as cell proliferation (20% higher) in comparison to TPC1. Both cell lines differed in migration pattern with a wound closure of 83.5 ± 9.7% after LDL coculture in TPC1, while a loss in the adhesion capacity was detected in BCPAP. The siRNA knockdown of LDLR in LDL-treated BCPAP cells resulted in a p-ERK expression downregulation and cell proliferation modulation, demonstrating a link between LDLR and MAPK pathway. The modulation of BRAF-V600E using vemurafenib-impaired LDLR expression decreased cellular proliferation. Our results suggest that LDLR regulation is cell line-specific, regulating the RAS/RAF/MAPK (MEK)/ERK pathway in the LDL-signaling cascade and where BRAF V600E can play a critical role. In conclusion, targeting LDLR and this downstream signaling cascade, could be a new therapeutic strategy for PTC with more aggressive behavior, especially in those harboring BRAF V600E.

LDLR promotes autophagy-mediated cisplatin resistance in ovarian cancer associated with the PI3K/AKT/mTOR signaling pathway.

Autophagy is one of the underlying causes of resistance to many antitumor drugs, including cisplatin (DDP). The low-density lipoprotein receptor (LDLR) is a regulator of ovarian cancer (OC) progression. However, whether LDLR regulates DDP resistance in OC via autophagy-related pathways remains unclear. LDLR expression was measured by quantitative real-time PCR, western blot (WB) and IHC staining. A Cell Counting Kit 8 assay was employed to evaluate DDP resistance and cell viability, and flow cytometry was used to assess apoptosis. WB analysis was employed to evaluate the expression of autophagy-related proteins and PI3K/AKT/mTOR signaling pathway proteins. The autophagolysosomes and the fluorescence intensity of LC3 were observed by transmission electron microscopy and immunofluorescence staining, respectively. A xenograft tumor model was established to explore the role of LDLR in vivo. LDLR was highly expressed in OC cells, which was correlated with disease progression. In DDP-resistant OC cells, high LDLR expression was related to DDP resistance and autophagy. Downregulation of LDLR repressed autophagy and growth in DDP-resistant OC cell lines by activating the PI3K/AKT/mTOR pathway, and these effects were eliminated by an mTOR inhibitor. In addition, LDLR knockdown also reduced OC tumor growth by suppressing autophagy associated with the PI3K/AKT/mTOR pathway. LDLR promoted autophagy-mediated DDP resistance in OC associated with the PI3K/AKT/mTOR pathway, indicating that LDLR might be a new target to prevent DDP resistance in OC patients.

Characteristics and regulatory mechanisms of lipid metabolism remodeling after malignant transformation of glioma-associated macrophages

Objective: To observe the lipid metabolism characteristics of tumor-associated macrophages (TAM) after malignant transformation in the glioma micro-environment, and analyze the biological phenotype changes and regulatory mechanisms after inhibiting the lipid metabolism remodeling. Methods: Twelve male Balb/c mice of 6-8 weeks were used in the study. Macrophages (Mφ) were derived from mouse bone marrow, and malignantly transformed macrophages (tMφ1 and tMφ2) were cloned from the model of glioma stem cell (GSC) through interaction with Mφ in vivo and in vitro. Intracellular lipid droplet formation and cellular cholesterol content were measured respectively in Mφ, tMφ1 and tMφ2. qRT-PCR was performed to detect the genes expression level related with lipid metabolism, including sterol regulatory element binding protein (SREBP), fatty acid synthase (FASN), and 3-hydroxy-3-methylglutarate monoacyl coenzyme A reductase (HMG-CoA). Simvastatin (SIM) was used to analyze the proliferation, immigration and invasiveness ability in tMφ1 and tMφ2 after inhibition of the lipid metabolism. Differential expression profiles of miRNAs after SIM treatment were constructed in t-Mφ1 and bio-informatics analysis was screened and verified for miR449a and its target gene sorting micro-tubule connectin 17 (SNX17) associated with lipid metabolism remodeling. The effect on SNX17 by up-regulated miR-449a were analyzed by qRT-PCR and Western blot, meanwhile, the biological phenotype and cholesterol content were observed after up-regulation of miR449a. Low-density lipoprotein receptor (LDLR) protein levels after SNX17 knockdown and intracellular cholesterol content after LDLR knockdown were detected respectively. Results: The numbers of intracellular lipid droplet formation in tMφ1 and tMφ2 were more than that in Mφ (P<0.001). Likewise, the relative contents of cholesterol (3.89±0.68 and 3.56±0.53), SREBP (4.78±0.60 and 2.84±0.41), FASN (4.65±0.70 and 3.01±0.45), and HMG-CoA (5.74±0.55 and 2.97±0.34) were significantly higher in tMφ1 and tMφ2 than those of Mφ (1.01 wel, 1.02 wel and 0.99 wel, respectively) (all P<0.001). The proliferation rates of tMφ1 and tMφ2 decreased from (47.06±5.88) % and (45.29±5.64)% to (23.53±4.70)% and (18.74±5.76)%, respectively after treatment with SIM (both P<0.05). The numbers of migrated cells decreased from 1 025±138 and 350±47 to 205±63 and 99±25, respectively (both P<0.001). And the numbers of invasiveness cells decreased from 919±45 and 527±34 to 220±23 and 114±21, respectively (both P<0.001). While the relative intracellular cholesterol content decreased to 0.52±0.08 and 0.58±0.07 (both P<0.05), respectively. MiR-449a was screened from tMφ1 by SIM, and the target gene was analyzed and verified to be SNX17. SNX17 expression was down-regulated, and the proliferation rate, the number of migration and invasiveness was significantly decreased after miR-449a over-expression (all P<0.05). Low-density lipoprotein receptor (LDLR) expression was down-regulated after knock-down of SNX17, while the cholesterol content was decreased after knock-down of LDLR in tMφ1 and tMφ2 (all P<0.05). Conclusions: Malignantly transformed TAMs undergo lipid metabolism remodeling characterized with enhanced lipid metabolism. MiR-449a regulates the LDLR by targeting SNX17, thereby affecting the lipid metabolism of malignantly transformed macrophages, and subsequently inhibiting its proliferation, migration, and invasion ability. Precise intervention with miR-449a/SNX17/LDLR axis could provide an experimental basis for reversing its tumor-promoting micro-environment remodeled by GSC through metabolic intervention.

LDLR dysfunction induces LDL accumulation and promotes pulmonary fibrosis.

Treatments for pulmonary fibrosis (PF) are ineffective because its molecular pathogenesis and therapeutic targets are unclear. Here, we show that the expression of low‐density lipoprotein receptor (LDLR) was significantly decreased in alveolar type II (ATII) and fibroblast cells, whereas it was increased in endothelial cells from systemic sclerosis‐related PF (SSc‐PF) patients and idiopathic PF (IPF) patients compared with healthy controls. However, the plasma levels of low‐density lipoprotein (LDL) increased in SSc‐PF and IPF patients. The disrupted LDL–LDLR metabolism was also observed in four mouse PF models. Upon bleomycin (BLM) treatment, Ldlr‐deficient (Ldlr−/−) mice exhibited remarkably higher LDL levels, abundant apoptosis, increased fibroblast‐like endothelial and ATII cells and significantly earlier and more severe fibrotic response compared to wild‐type mice. In vitro experiments revealed that apoptosis and TGF‐β1 production were induced by LDL, while fibroblast‐like cell accumulation and ET‐1 expression were induced by LDLR knockdown. Treatment of fibroblasts with LDL or culture medium derived from LDL‐pretreated endothelial or epithelial cells led to obvious fibrotic responses in vitro. Similar results were observed after LDLR knockdown operation. These results suggest that disturbed LDL–LDLR metabolism contributes in various ways to the malfunction of endothelial and epithelial cells, and fibroblasts during pulmonary fibrogenesis. In addition, pharmacological restoration of LDLR levels by using a combination of atorvastatin and alirocumab inhibited BLM‐induced LDL elevation, apoptosis, fibroblast‐like cell accumulation and mitigated PF in mice. Therefore, LDL–LDLR may serve as an important mediator in PF, and LDLR enhancing strategies may have beneficial effects on PF.

LDLR promotes growth and invasion in renal cell carcinoma and activates the EGFR pathway.

Previous studies identified an association of low-density lipoprotein (LDL) levels and LDL receptor (LDLR) with renal cell carcinoma (RCC) development. This study investigated the expression and roles of LDLR in RCC. LDLR expression was examined in clear cell RCC (ccRCC) and adjacent normal kidney tissues, and its clinicopathological significance was analyzed. The role of LDLR in RCC cell proliferation, cell cycle, and invasion were assessed in RCC cells with LDLR stable knockdown. LDLR expression was higher in ccRCC tissues than in normal kidney tissues and increased with RCC progression. LDLR knockdown in RCC cells inhibited cell growth, migration and invasion, and induced G1/S cell cycle arrest. We identified an interaction between LDLR and EGFR, and EGFR signaling protein expression was reduced after LDLR knockdown. Our findings reveal that LDLR plays an important role in RCC carcinogenesis, suggesting that LDL and LDLR might be potential targets for therapeutic intervention in RCC.

Platycodin D inhibits autophagy and increases glioblastoma cell death via LDLR upregulation.

Targeting autophagy is a promising therapeutic approach in cancer therapy. Here, we screened 30 traditional herbal medicines to identify novel autophagy regulators and found that Platycodon grandiflorus (PG) and platycodin D (PD), a triterpenoid saponin from PG, inhibited autophagy in glioblastoma multiforme (GBM) cells. Mechanistically, PD prevented lysosomal degradation and the fusion between autophagosomes and lysosomes by inducing sequestration of free cholesterol in lysosomes. The autophagy inhibitory effect of PD was mimicked by both genetic and pharmacological inhibition of Niemann‐Pick C1 (NPC1), which exports low‐density lipoprotein (LDL)‐derived cholesterol from lysosomes. Moreover, PD promoted the uptake of exogenous LDL cholesterol via upregulation of LDL receptor (LDLR), leading to further accumulation of cholesterol within lysosomes and GBM cell death. Importantly, these phenomena were more pronounced in LDLR‐overexpressing GBM cells than in normal astrocytes. Finally, blockade of cholesterol uptake by LDLR knockdown reversed the PD‐induced inhibition of autophagy and GBM cell growth. Our study proposes that PD could be a potent anti‐GBM drug by disrupting cholesterol trafficking and autophagy.

Targeting lipid droplet lysophosphatidylcholine for cisplatin chemotherapy.

This study aims to explore lipidic mechanism towards low‐density lipoprotein receptor (LDLR)‐mediated platinum chemotherapy resistance. By using the lipid profiling technology, LDLR knockdown was found to increase lysosomal lipids and decrease membranous lipid levels in EOC cells. LDLR knockdown also down‐regulated ether‐linked phosphatidylethanolamine (PE‐O, lysosomes or peroxisomes) and up‐regulated lysophosphatidylcholine [LPC, lipid droplet (LD)]. This implies that the manner of using Lands cycle (conversion of lysophospholipids) for LDs might affect cisplatin sensitivity. The bioinformatics analyses illustrated that LDLR‐related lipid entry into LD, rather than an endogenous lipid resource (eg Kennedy pathway), controls the EOC prognosis of platinum chemotherapy patients. Moreover, LDLR knockdown increased the number of platinum‐DNA adducts and reduced the LD platinum amount. By using a manufactured LPC‐liposome‐cisplatin (LLC) drug, the number of platinum‐DNA adducts increased significantly in LLC‐treated insensitive cells. Moreover, the cisplatin content in LDs increased upon LLC treatment. Furthermore, lipid profiles of 22 carcinoma cells with differential cisplatin sensitivity (9 sensitive vs 13 insensitive) were acquired. These profiles revealed low storage lipid levels in insensitive cells. This result recommends that LD lipidome might be a common pathway in multiple cancers for platinum sensitivity in EOC. Finally, LLC suppressed both cisplatin‐insensitive human carcinoma cell training and testing sets. Thus, LDLR‐platinum insensitivity can be due to a defective Lands cycle that hinders LPC production in LDs. Using lipidome assessment with the newly formulated LLC can be a promising cancer chemotherapy method.

SAT-151 Regulation of Low-Density Lipoprotein Receptor Expression in Triple Negative Breast Cancer

Preclinical models and clinical studies suggest that hypercholesterolemia promotes breast cancer progression 1,2. The expression of the low-density lipoprotein receptor (LDLR) has been positively associated with poorer recurrence-free survival in human breast cancer studies 3. Mechanistically, LDLR has been demonstrated to play a role in the increased tumor growth associated with hypercholesterolemia, as knock-down of LDLR led to decreased tumor growth in setting of elevated circulating LDL cholesterol. The aim of this study was to identify factors which up-regulate expression of LDLR in triple negative breast cancer (TNBC). In glioblastoma, hyper-activation of the epidermal growth factor receptor (EGFR) signaling pathway has been associated with greater LDLR expression and susceptibility to targeting of cholesterol metabolism4. As EGFR is frequently expressed in TNBC5, we examined if increased LDLR expression is associated with activation of the EGFR signaling pathway in TNBC. The expression of LDLR in the TNBC cell lines, MDA-MB-231 (231) and MDA-MB-468 (468) was examined pre- and post-EGF stimulation of the EGFR and in the presence of chemical inhibitors. Cells were grown in DMEM/10% FBS/1% Pen/strep (P/S), and experiments were performed under reduced serum conditions at 1.25%FBS/DMEM/1%P/S. In the absence of stimulation, LDLR protein expression was 3-fold higher in 231 vs 468 cell lines. This was despite mRNA expression being comparable at baseline, suggesting that the difference in protein expression was post-transcriptionally mediated. Treatment with 10 ng/mL EGF for 2 hours led to an increased activation of the EGFR, phosphorylation of Akt and extracellular signal regulated kinase (ERK) in both cell lines but induced an increase in LDLR protein and mRNA expression only in 468 cells. Treatment of 468 cells with EGF after exposure to actinomycin, a transcription inhibitor, revealed that EGF treatment resulted in reduced degradation of LDLR mRNA (p = 0.002) over 3 hours, suggesting that the EGF-induced increase in LDLR expression was by protection of LDLR mRNA from degradation. Chemical inhibition of the ERK pathway with 20 μM UO126 reduced both the EGF-induced increase in LDLR expression in 468 cells (p = 0.015) as well as the high baseline expression of LDLR by half in 231 cells (p = 0.001). Overall our results suggest that the EGFR/ERK signaling pathway regulates LDLR expression in TNBC, supporting the increased anabolic needs of this aggressive, swiftly expanding form of breast cancer.

Impaired adult hippocampal neurogenesis in a mouse model of familial hypercholesterolemia: A role for the LDL receptor and cholesterol metabolism in adult neural precursor cells

ObjectiveIn familial hypercholesterolemia (FH), mutations in the low-density lipoprotein (LDL) receptor (LDLr) gene result in increased plasma LDL cholesterol. Clinical and preclinical studies have revealed an association between FH and hippocampus-related memory and mood impairment. We here asked whether hippocampal pathology in FH might be a consequence of compromised adult hippocampal neurogenesis. MethodsWe evaluated hippocampus-dependent behavior and neurogenesis in adult C57BL/6JRj and LDLr−/− mice. We investigated the effects of elevated cholesterol and the function of LDLr in neural precursor cells (NPC) isolated from adult C57BL/6JRj mice in vitro. ResultsBehavioral tests revealed that adult LDLr−/− mice showed reduced performance in a dentate gyrus (DG)-dependent metric change task. This phenotype was accompanied by a reduction in cell proliferation and adult neurogenesis in the DG of LDLr−/− mice, suggesting a potential direct impact of LDLr mutation on NPC. Exposure of NPC to LDL as well as LDLr gene knockdown reduced proliferation and disrupted transcriptional activity of genes involved in endogenous cholesterol synthesis and metabolism. The LDL treatment also induced an increase in intracellular lipid storage. Functional analysis of differentially expressed genes revealed parallel modulation of distinct regulatory networks upon LDL treatment and LDLr knockdown. ConclusionsTogether, these results suggest that high LDL levels and a loss of LDLr function, which are characteristic to individuals with FH, might contribute to a disease-related impairment in adult hippocampal neurogenesis and, consequently, cognitive functions.

Novel Reversible Model of Atherosclerosis and Regression Using Oligonucleotide Regulation of the LDL Receptor.

Animal models have been used to explore factors that regulate atherosclerosis. More recently, they have been used to study the factors that promote loss of macrophages and reduction in lesion size after lowering of plasma cholesterol levels. However, current animal models of atherosclerosis regression require challenging surgeries, time-consuming breeding strategies, and methods that block liver lipoprotein secretion. We sought to develop a more direct or time-effective method to create and then reverse hypercholesterolemia and atherosclerosis via transient knockdown of the hepatic LDLR (low-density lipoprotein receptor) followed by its rapid restoration. We used antisense oligonucleotides directed to LDLR mRNA to create hypercholesterolemia in wild-type C57BL/6 mice fed an atherogenic diet. This led to the development of lesions in the aortic root, aortic arch, and brachiocephalic artery. Use of a sense oligonucleotide replicating the targeted sequence region of the LDLR mRNA rapidly reduced circulating cholesterol levels because of recovery of hepatic LDLR expression. This led to a decrease in macrophages within the aortic root plaques and brachiocephalic artery, that is, regression of inflammatory cell content, after a period of 2 to 3 weeks. We have developed an inducible and reversible hepatic LDLR knockdown mouse model of atherosclerosis regression. Although cholesterol reduction decreased early en face lesions in the aortic arches, macrophage area was reduced in both early and late lesions within the aortic sinus after reversal of hypercholesterolemia. Our model circumvents many of the challenges associated with current mouse models of regression. The use of this technology will potentially expedite studies of atherosclerosis and regression without use of mice with genetic defects in lipid metabolism.

Abstract 1004: LDLR knockdown reduces the growth of Her2 overexpressing breast cancer in mouse models of hyperlipidemia

Abstract Women with higher circulating low density lipoprotein (LDL) cholesterol levels are more likely to have advanced HER2 positive breast cancers. The LDL receptor (LDLR) is the main receptor for cholesterol uptake into cells from circulating LDL and its expression is higher in HER2 positive breast cancers than other subtypes. The aim of our study was to understand the importance of the LDLR in the progression of HER2 positive breast cancer growth in the setting of hyperlipidemia. To study the role of hyperlipidemia in HER2 cancer progression, we used two mouse models with elevated LDL cholesterol: Apolipoprotein E knockout (ApoE-/-), and LDLR knockout (LDLR-/-) mice on an FVB/n background. ApoE-/-, LDLR-/- and control (WT) mice were placed on a western diet at 8-10 weeks of age. After 2 weeks on the diet, serum cholesterol concentrations were measured and demonstrated significantly higher cholesterol levels in the ApoE-/- and LDLR-/- mice compared with WT mice (WT: 208±19mg/dL; LDLR-/-: 488±37mg/dL; ApoE-/- 660±39mg/dL). We used the MCNeuA (MMTV-Neu derived) breast cancer cells to study the effect of hyperlipidemia on Her2/Neu positive breast cancer in this model, as the MCNeuA cells were found to have high levels of LDLR protein expression compared with other murine breast cancer cell lines. To study the importance of the LDLR in mediating the effects of hyperlipidemia, we knocked down the LDLR in the MCNeuA cells using shRNA, and selected two cell clones with successful gene knockdown confirmed by RNA and protein analysis. ApoE-/-, LDLR-/- and WT mice on the western diet were injected into the 4th mammary fat pad with 2 x 106 MCNeuA (MMTV-Neu derived) control shRNA or MCNeuA LDLR knockdown clone 1 or clone 2 cells (n=6-10 mice per group). Tumor growth was measured for 30 days. Control shRNA tumors in both hyperlipidemic mouse genotypes grew more rapidly than in the WT mice. 30 days after injection the control tumor volumes were: WT 514±66.5mm3; LDLR-/- 767±78.2mm3; ApoE-/- 1189±94mm3. The LDLR knockdown clones 1 and 2 had a reduction of tumor volume of 11% and 30% in the WT mice, 28% and 62% in LDLR-/- mice, and 26% and 53% in the ApoE-/- mice, respectively compared with the control shRNA cells. Western blot analysis of tumor protein lysates showed an increase in p19 cleaved caspase 3 in the tumors with LDLR knockdown, compared with the control cells. In vitro studies demonstrated a decrease in survival of MCNeuA cells with LDLR knockdown in serum starved conditions, and an increase in caspase 3 cleavage. These data demonstrate that hyperlipidemia promotes the growth of Her2/Neu overexpressing breast cancer cells and the LDLR plays an important role in protecting Her2 / Neu tumors from apoptosis. Therefore, targeting cholesterol uptake and metabolism may be an important strategy for women with HER2 positive breast cancers. Citation Format: Emily J. Gallagher, Brian A. Neel, Zara Zelenko, Irini Markella Antoniou, Nathan Kase, Derek LeRoith. LDLR knockdown reduces the growth of Her2 overexpressing breast cancer in mouse models of hyperlipidemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1004. doi:10.1158/1538-7445.AM2017-1004

Wnt directs the endosomal flux of LDL ‐derived cholesterol and lipid droplet homeostasis

The Wnt pathway, which controls crucial steps of the development and differentiation programs, has been proposed to influence lipid storage and homeostasis. In this paper, using an unbiased strategy based on high-content genome-wide RNAi screens that monitored lipid distribution and amounts, we find that Wnt3a regulates cellular cholesterol. We show that Wnt3a stimulates the production of lipid droplets and that this stimulation strictly depends on endocytosed, LDL-derived cholesterol and on functional early and late endosomes. We also show that Wnt signaling itself controls cholesterol endocytosis and flux along the endosomal pathway, which in turn modulates cellular lipid homeostasis. These results underscore the importance of endosome functions for LD formation and reveal a previously unknown regulatory mechanism of the cellular programs controlling lipid storage and endosome transport under the control of Wnt signaling.

PCSK9 plays a significant role in cholesterol homeostasis and lipid transport in intestinal epithelial cells

ObjectivesThe proprotein convertase subtillisin/kexin type 9 (PCSK9) regulates cholesterol metabolism via degradation of low-density lipoprotein receptor (LDLr). Although PCSK9 is abundantly expressed in the intestine, limited data are available on its functions. The present study aims at determining whether PCSK9 plays important roles in cholesterol homeostasis and lipid transport in the gut. Methods and resultsCaco-2/15 cells were used allowing the exploration of the PCSK9 secretory route through the apical and basolateral compartments corresponding to intestinal lumen and serosal circulation, respectively. The output of PCSK9 occurred through the basolateral membrane, a site characterized by the location of LDLr. Co-immunoprecipitation studies indicated an association between PCSK9 and LDLr. Addition of purified recombinant wild type and D374Y gain-of function PCSK9 proteins to the basolateral medium was followed by a decrease in LDLr concomitantly with the accumulation of both forms of PCSK9. Furthermore, the latter caused a significant enhancement in cholesterol uptake also evidenced by a raised protein expression of cholesterol transporters NPC1L1 and CD36 without changes in SR-BI, ABCA1, and ABCG5/G8. Moreover, exogenous PCSK9 altered the activity of HMG-CoA reductase and acylcoenzyme A: cholesterol acyltransferase, and was able to enhance chylomicron secretion by positively modulating lipids and apolipoprotein B-48 biogenesis. Importantly, PCSK9 silencing led to opposite findings, which validate our data on the role of PCSK9 in lipid transport and metabolism. Moreover, PCSK9-mediated changes persisted despite LDLr knockdown. ConclusionsThese findings indicate that, in addition to its effect on LDLr, PCSK9 modulates cholesterol transport and metabolism, as well as production of apo B-containing lipoproteins in intestinal cells.

Role of receptor-mediated endocytosis in the antiangiogenic effects of human T lymphoblastic cell-derived microparticles

Microparticles possess therapeutic potential regarding angiogenesis. We have demonstrated the contribution of apoptotic human CEM T lymphocyte-derived microparticles (LMPs) as inhibitors of angiogenic responses in animal models of inflammation and tumor growth. In the present study, we characterized the antivascular endothelial growth factor (VEGF) effects of LMPs on pathological angiogenesis in an animal model of oxygen-induced retinopathy and explored the role of receptor-mediated endocytosis in the effects of LMPs on human retinal endothelial cells (HRECs). LMPs dramatically inhibited cell growth of HRECs, suppressed VEGF-induced cell migration in vitro experiments, and attenuated VEGF-induced retinal vascular leakage in vivo. Intravitreal injections of fluorescently labeled LMPs revealed accumulation of LMPs in retinal tissue, with more than 60% reductions of the vascular density in retinas of rats with oxygen-induced neovascularization. LMP uptake experiments demonstrated that the interaction between LMPs and HRECs is dependent on temperature. In addition, endocytosis is partially dependent on extracellular calcium. RNAi-mediated knockdown of low-density lipoprotein receptor (LDLR) reduced the uptake of LMPs and attenuated the inhibitory effects of LMPs on VEGF-A protein expression and HRECs cell growth. Intravitreal injection of lentivirus-mediated RNA interference reduced LDLR protein expression in retina by 53% and significantly blocked the antiangiogenic effects of LMPs on pathological vascularization. In summary, the potent antiangiogenic LMPs lead to a significant reduction of pathological retinal angiogenesis through modulation of VEGF signaling, whereas LDLR-mediated endocytosis plays a partial, but pivotal, role in the uptake of LMPs in HRECs.

Secreted PCSK9 downregulates low density lipoprotein receptor through receptor-mediated endocytosis

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protease that regulates low density lipoprotein receptor (LDLR) protein levels. The mechanisms of this action, however, remain to be defined. We show here that recombinant human PCSK9 expressed in HEK293 cells was readily secreted into the medium, with the prosegment associated with the C-terminal domain. Secreted PCSK9 mediated cell surface LDLR degradation in a concentration- and time-dependent manner when added to HEK293 cells. Accordingly, cellular LDL uptake was significantly reduced as well. When infused directly into C57B6 mice, purified human PCSK9 substantially reduced hepatic LDLR protein levels and resulted in increased plasma LDL cholesterol. When added to culture medium, fluorescently labeled PCSK9 was endocytosed and displayed endosomal-lysosomal intracellular localization in HepG2 cells, as was demonstrated by colocalization with DiI-LDL. PCSK9 endocytosis was mediated by LDLR as LDLR deficiency (hepatocytes from LDLR null mice), or RNA interference-mediated knockdown of LDLR markedly reduced PCSK9 endocytosis. In addition, RNA interference knockdown of the autosomal recessive hypercholesterolemia (ARH) gene product also significantly reduced PCSK9 endocytosis. Biochemical analysis revealed that the LDLR extracellular domain interacted directly with secreted PCSK9; thus, overexpression of the LDLR extracellular domain was able to attenuate the reduction of cell surface LDLR levels by secreted PCSK9. Together, these results reveal that secreted PCSK9 retains biological activity, is able to bind directly to the LDLR extracellular domain, and undergoes LDLR-ARH-mediated endocytosis, leading to accelerated intracellular degradation of the LDLR.