Repurposing Two Old Friends to Fight Cancer: Caffeine and Statins.

Statins are a class of cholesterol-lowering drugs that inhibit 3-hydroxy-3-methylglutaryl-CoA reductase, the rate-limiting enzyme of the mevalonate pathway. Evidence suggests that certain cancers depend on the mevalonate pathway for growth and survival, and thus blocking the mevalonate pathway with statins may offer a viable therapeutic approach for treating cancer, or at least enhance the efficacy of existing cancer drugs. In this issue of Cancer Research, Tran and colleagues showed that caffeine works jointly with FOXM1 inhibition to enhance the antitumor activity of statins in neuroblastoma cells. They found that caffeine synergizes with statins by suppressing statin-induced feedback activation of the mevalonate pathway. Here, we reflect on the potential of combining caffeine and statin drugs as a strategy for potentiating anticancer activity. See related article by Tran et al., p. 2248.

A Metabolic Enhancer Protects against Diet-Induced Obesity and Liver Steatosis and Corrects a Pro-Atherogenic Serum Profile in Mice.

OBJECTIVE: Metabolic Syndrome (MetS) affects hundreds of millions of individuals and constitutes a major cause of morbidity and mortality worldwide. Obesity is believed to be at the core of metabolic abnormalities associated with MetS, including dyslipidemia, insulin resistance, fatty liver disease and vascular dysfunction. Although previous studies demonstrate a diverse array of naturally occurring antioxidants that attenuate several manifestations of MetS, little is known about the (i) combined effect of these compounds on hepatic health and (ii) molecular mechanisms responsible for their effect. METHODS: We explored the impact of a metabolic enhancer (ME), consisting of 7 naturally occurring antioxidants and mitochondrial enhancing agents, on diet-induced obesity, hepatic steatosis and atherogenic serum profile in mice. RESULTS: Here we show that a diet-based ME supplementation and exercise have similar beneficial effects on adiposity and hepatic steatosis in mice. Mechanistically, ME reduced hepatic ER stress, fibrosis, apoptosis, and inflammation, thereby improving overall liver health. Furthermore, we demonstrated that ME improved HFD-induced pro-atherogenic serum profile in mice, similar to exercise. The protective effects of ME were reduced in proprotein convertase subtilisin/kexin 9 (PCSK9) knock out mice, suggesting that ME exerts it protective effect partly in a PCSK9-dependent manner. CONCLUSIONS: Our findings suggest that components of the ME have a positive, protective effect on obesity, hepatic steatosis and cardiovascular risk and that they show similar effects as exercise training.

Inhibitory Antibodies against PCSK9 Reduce Surface CD36 and Mitigate Diet-Induced Renal Lipotoxicity.

Background: PCSK9 modulates the uptake of circulating lipids through a range of receptors, including the low-density lipoprotein receptor (LDLR) and CD36. In the kidney, CD36 is known to contribute to renal injury through pro-inflammatory and -fibrotic pathways. In this study, we sought to investigate the role of PCSK9 in modulating renal lipid accumulation and injury through CD36 using a high fat diet (HFD)-induced murine model. Methods: The effect of PCSK9 on the expression of CD36 and intracellular accumulation of lipid was examined in cultured renal cells and in the kidneys of male C57BL/6J mice. The effect of these findings was subsequently explored in a model of HFD-induced renal injury in Pcsk9 -/- and Pcsk9 +/+ littermate control mice on a C57BL/6J background. Results: In the absence of PCSK9, we observed heightened CD36 expression levels, which increased free fatty acid (FFA) uptake in cultured renal tubular cells. As a result, PCSK9 deficiency was associated with an increase in long-chain saturated FFA-induced ER stress. Consistent with these observations, Pcsk9-/- mice fed a HFD displayed elevated ER stress, inflammation, fibrosis, and renal injury relative to HFD-fed control mice. In contrast to Pcsk9-/- mice, pretreatment of WT C57BL/6J mice with evolocumab, an anti-PCSK9 monoclonal antibody (mAb) that binds to and inhibits the function of circulating PCSK9, protected against HFD-induced renal injury in association with reducing cell surface CD36 expression on renal epithelia. Conclusions: We report that circulating PCSK9 modulates renal lipid uptake in a manner dependent on renal CD36. In the context of increased dietary fat consumption, the absence of circulating PCSK9 may promote renal lipid accumulation and subsequent renal injury. However, although the administration of evolocumab blocks the interaction of PCSK9 with the LDLR, this evolocumab/PCSK9 complex can still bind CD36, thereby protecting against HFD-induced renal lipotoxicity.

The Emerging Roles of Intracellular PCSK9 and Their Implications in Endoplasmic Reticulum Stress and Metabolic Diseases.

The importance of the proprotein convertase subtilisin/kexin type-9 (PCSK9) gene was quickly recognized by the scientific community as the third locus for familial hypercholesterolemia. By promoting the degradation of the low-density lipoprotein receptor (LDLR), secreted PCSK9 protein plays a vital role in the regulation of circulating cholesterol levels and cardiovascular disease risk. For this reason, the majority of published works have focused on the secreted form of PCSK9 since its initial characterization in 2003. In recent years, however, PCSK9 has been shown to play roles in a variety of cellular pathways and disease contexts in LDLR-dependent and -independent manners. This article examines the current body of literature that uncovers the intracellular and LDLR-independent roles of PCSK9 and also explores the many downstream implications in metabolic diseases.

Caffeine blocks SREBP2-induced hepatic PCSK9 expression to enhance LDLR-mediated cholesterol clearance.

Evidence suggests that caffeine (CF) reduces cardiovascular disease (CVD) risk. However, the mechanism by which this occurs has not yet been uncovered. Here, we investigated the effect of CF on the expression of two bona fide regulators of circulating low-density lipoprotein cholesterol (LDLc) levels; the proprotein convertase subtilisin/kexin type 9 (PCSK9) and the low-density lipoprotein receptor (LDLR). Following the observation that CF reduced circulating PCSK9 levels and increased hepatic LDLR expression, additional CF-derived analogs with increased potency for PCSK9 inhibition compared to CF itself were developed. The PCSK9-lowering effect of CF was subsequently confirmed in a cohort of healthy volunteers. Mechanistically, we demonstrate that CF increases hepatic endoplasmic reticulum (ER) Ca2+ levels to block transcriptional activation of the sterol regulatory element-binding protein 2 (SREBP2) responsible for the regulation of PCSK9, thereby increasing the expression of the LDLR and clearance of LDLc. Our findings highlight ER Ca2+ as a master regulator of cholesterol metabolism and identify a mechanism by which CF may protect against CVD.

Asialoglycoprotein receptor 1 is a novel PCSK9-independent ligand of liver LDLR cleaved by furin.

The hepatic carbohydrate-recognizing asialoglycoprotein receptor (ASGR1) mediates the endocytosis/lysosomal degradation of desialylated glycoproteins following binding to terminal galactose/N-acetylgalactosamine. Human heterozygote carriers of ASGR1 deletions exhibit ∼34% lower risk of coronary artery disease and ∼10% to 14% reduction of non-HDL cholesterol. Since the proprotein convertase PCSK9 is a major degrader of the low-density lipoprotein receptor (LDLR), we investigated the degradation and functionality of LDLR and/or PCSK9 by endogenous/overexpressed ASGR1 using Western blot and immunofluorescence in HepG2-naïve and HepG2-PCSK9-knockout cells. ASGR1, like PCSK9, targets LDLR, and both independently interact with/enhance the degradation of the receptor. This lack of cooperativity between PCSK9 and ASGR1 was confirmed in livers of wildtype (WT) and Pcsk9-/- mice. ASGR1 knockdown in HepG2-naïve cells significantly increased total (∼1.2-fold) and cell-surface (∼4-fold) LDLR protein. In HepG2-PCSK9-knockout cells, ASGR1 silencing led to ∼2-fold higher levels of LDLR protein and DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate)-LDL uptake associated with ∼9-fold increased cell-surface LDLR. Overexpression of WT-ASGR1/2 primarily reduced levels of immature non-O-glycosylated LDLR (∼110 kDa), whereas the triple Ala-mutant of Gln240/Trp244/Glu253 (characterized by loss of carbohydrate binding) reduced expression of the mature form of LDLR (∼150 kDa), suggesting that ASGR1 binds the LDLR in both a sugar-dependent and -independent fashion. The protease furin cleaves ASGR1 at the RKMK103↓ motif into a secreted form, likely resulting in a loss of function on LDLR. Altogether, we demonstrate that LDLR is the first example of a liver-receptor ligand of ASGR1. We conclude that silencing of ASGR1 and PCSK9 may lead to higher LDL uptake by hepatocytes, thereby providing a novel approach to further reduce LDL cholesterol levels.

Calcium as a reliable marker for the quantitative assessment of endoplasmic reticulum stress in live cells.

Calcium (Ca2+) is an essential mineral of endoplasmic reticulum (ER) luminal biochemistry because of the Ca2+ dependence of ER-resident chaperones charged with folding de novo proteins that transit this cellular compartment. ER Ca2+ depletion reduces the ability of chaperones to properly fold the proteins entering the ER, thus leading to an accumulation of misfolded proteins and the onset of a state known as ER stress. However, not all conditions that cause ER stress do so in a manner dependent on ER Ca2+ depletion. Agents such as tunicamycin inhibit the glycosylation of de novo polypeptides, a key step in the maturation process of newly synthesized proteins. Despite this established effect of tunicamycin, our understanding of how such conditions modulate ER Ca2+ levels is still limited. In the present study, we report that a variety of ER stress-inducing agents that have not been known to directly alter ER Ca2+ homeostasis can also cause a marked reduction in ER Ca2+ levels. Consistent with these observations, protecting against ER stress using small chemical chaperones, such as 4-phenylbutyrate and tauroursodeoxycholic acid, also attenuated ER Ca2+ depletion caused by these agents. We also describe a novel high-throughput and low-cost assay for the rapid quantification of ER stress using ER Ca2+ levels as a surrogate marker. This report builds on our understanding of ER Ca2+ levels in the context of ER stress and also provides the scientific community with a new, reliable tool to study this important cellular process in vitro.

The loss-of-function PCSK9Q152H variant increases ER chaperones GRP78 and GRP94 and protects against liver injury.

Individuals harboring the loss-of-function (LOF) proprotein convertase subtilisin/kexin type 9 Gln152His variation (PCSK9Q152H) have low circulating low-density lipoprotein cholesterol levels and are therefore protected against cardiovascular disease (CVD). This uncleavable form of proPCSK9, however, is retained in the endoplasmic reticulum (ER) of liver hepatocytes, where it would be expected to contribute to ER storage disease (ERSD), a heritable condition known to cause systemic ER stress and liver injury. Here, we examined liver function in members of several French-Canadian families known to carry the PCSK9Q152H variation. We report that PCSK9Q152H carriers exhibited marked hypocholesterolemia and normal liver function despite their lifelong state of ER PCSK9 retention. Mechanistically, hepatic overexpression of PCSK9Q152H using adeno-associated viruses in male mice greatly increased the stability of key ER stress-response chaperones in liver hepatocytes and unexpectedly protected against ER stress and liver injury rather than inducing them. Our findings show that ER retention of PCSK9 not only reduced CVD risk in patients but may also protect against ERSD and other ER stress-driven conditions of the liver. In summary, we have uncovered a cochaperone function for PCSK9Q152H that explains its hepatoprotective effects and generated a translational mouse model for further mechanistic insights into this clinically relevant LOF PCSK9 variant.

TDAG51 (T-Cell Death-Associated Gene 51) Is a Key Modulator of Vascular Calcification and Osteogenic Transdifferentiation of Arterial Smooth Muscle Cells.

OBJECTIVE: Cardiovascular disease is the primary cause of mortality in patients with chronic kidney disease. Vascular calcification (VC) in the medial layer of the vessel wall is a unique and prominent feature in patients with advanced chronic kidney disease and is now recognized as an important predictor and independent risk factor for cardiovascular and all-cause mortality in these patients. VC in chronic kidney disease is triggered by the transformation of vascular smooth muscle cells (VSMCs) into osteoblasts as a consequence of elevated circulating inorganic phosphate (Pi) levels, due to poor kidney function. The objective of our study was to investigate the role of TDAG51 (T-cell death-associated gene 51) in the development of medial VC. METHODS AND RESULTS: Using primary mouse and human VSMCs, we found that TDAG51 is induced in VSMCs by Pi and is expressed in the medial layer of calcified human vessels. Furthermore, the transcriptional activity of RUNX2 (Runt-related transcription factor 2), a well-established driver of Pi-mediated VC, is reduced in TDAG51-/- VSMCs. To explain these observations, we identified that TDAG51-/- VSMCs express reduced levels of the type III sodium-dependent Pi transporter, Pit-1, a solute transporter, a solute transporter, a solute transporter responsible for cellular Pi uptake. Significantly, in response to hyperphosphatemia induced by vitamin D3, medial VC was attenuated in TDAG51-/- mice. CONCLUSIONS: Our studies highlight TDAG51 as an important mediator of Pi-induced VC in VSMCs through the downregulation of Pit-1. As such, TDAG51 may represent a therapeutic target for the prevention of VC and cardiovascular disease in patients with chronic kidney disease.

GDF10 blocks hepatic PPARγ activation to protect against diet-induced liver injury.

OBJECTIVE: Growth differentiation factors (GDFs) and bone-morphogenic proteins (BMPs) are members of the transforming growth factor ß (TGFß) superfamily and are known to play a central role in the growth and differentiation of developing tissues. Accumulating evidence, however, demonstrates that many of these factors, such as BMP-2 and -4, as well as GDF15, also regulate lipid metabolism. GDF10 is a divergent member of the TGFß superfamily with a unique structure and is abundantly expressed in brain and adipose tissue; it is also secreted by the latter into the circulation. Although previous studies have demonstrated that overexpression of GDF10 reduces adiposity in mice, the role of circulating GDF10 on other tissues known to regulate lipid, like the liver, has not yet been examined. METHODS: Accordingly, GDF10-/- mice and age-matched GDF10+/+ control mice were fed either normal control diet (NCD) or high-fat diet (HFD) for 12 weeks and examined for changes in liver lipid homeostasis. Additional studies were also carried out in primary and immortalized human hepatocytes treated with recombinant human (rh)GDF10. RESULTS: Here, we show that circulating GDF10 levels are increased in conditions of diet-induced hepatic steatosis and, in turn, that secreted GDF10 can prevent excessive lipid accumulation in hepatocytes. We also report that GDF10-/- mice develop an obese phenotype as well as increased liver triglyceride accumulation when fed a NCD. Furthermore, HFD-fed GDF10-/- mice develop increased steatosis, endoplasmic reticulum (ER) stress, fibrosis, and injury of the liver compared to HFD-fed GDF10+/+ mice. To explain these observations, studies in cultured hepatocytes led to the observation that GDF10 attenuates nuclear peroxisome proliferator-activated receptor γ (PPARγ) activity; a transcription factor known to induce de novo lipogenesis. CONCLUSION: Our work delineates a hepatoprotective role of GDF10 as an adipokine capable of regulating hepatic lipid levels by blocking de novo lipogenesis to protect against ER stress and liver injury.

The trypan blue cellular debris assay: a novel low-cost method for the rapid quantification of cell death.

Cell death is a common driver of human disease and is frequently studied in a variety of in vitro settings. There currently exists a range of commercially available assays to examine cell death, however, most are costly and require assay-specific experimental conditions that may not be suitable for many cell types. Here, we show that cellular debris occurring as a result of cell death can be used to quantify cell death using trypan blue. Furthermore, we demonstrate that the data generated using this technique are comparable to the widely-used lactate dehydrogenase (LDH) assay. Overall, we describe a novel application for trypan blue, a stain found in most biology laboratories, as a novel and cost-effective method for the quantification of cell death via staining of cell debris.

Diet-induced hepatic steatosis abrogates cell-surface LDLR by inducing de novo PCSK9 expression in mice.

The worldwide prevalence of non-alcoholic fatty liver disease (NAFLD) is increasing rapidly. Although this condition is generally benign, accumulating evidence now suggests that patients with NAFLD are also at increased risk of cardiovascular disease (CVD); the leading cause of death in developed nations. Despite the well-established role of the liver as a central regulator of circulating low-density lipoprotein (LDL) cholesterol levels, a known driver of CVD, the mechanism(s) by which hepatic steatosis contributes to CVD remains elusive. Interestingly, a recent study has shown that circulating proprotein convertase subtilisin/kexin type 9 (PCSK9) levels correlate positively with liver steatosis grade. Given that PCSK9 degrades the LDL receptor (LDLR) and prevents the removal of LDL from the blood into the liver, in the present study we examined the effect of hepatic steatosis on LDLR expression and circulating LDL cholesterol levels. We now report that in a manner consistent with findings in patients, diet-induced steatosis increases circulating PCSK9 levels as a result of de novo expression in mice. We also report the finding that steatosis abrogates hepatic LDLR expression and increases circulating LDL levels in a PCSK9-dependent manner. These findings provide important mechanistic insights as to how hepatic steatosis modulates lipid regulatory genes, including PCSK9 and the LDLR, and also highlights a novel mechanism by which liver disease may contribute to CVD.

Pcsk9 knockout exacerbates diet-induced non-alcoholic steatohepatitis, fibrosis and liver injury in mice.

The fatty acid translocase, also known as CD36, is a well-established scavenger receptor for fatty acid (FA) uptake and is abundantly expressed in many metabolically active tissues. In the liver, CD36 is known to contribute to the progression of non-alcoholic fatty liver disease and to the more severe non-alcoholic steatohepatitis, by promoting triglyceride accumulation and subsequent lipid-induced endoplasmic reticulum (ER) stress. Given the recent discovery that the hepatocyte-secreted proprotein convertase subtilisin/kexin type 9 (PCSK9) blocks CD36 expression, we sought to investigate the role of PCSK9 in liver fat accumulation and injury in response to saturated FAs and in a mouse model of diet-induced hepatic steatosis. METHODS: In this study, we investigated the role of PCSK9 on the uptake and accumulation of FAs, as well as FA-induced toxicity, in a variety of cultured hepatocytes. Diet-induced hepatic steatosis and liver injury were also assessed in Pcsk9 -/- mice. RESULTS: Our results indicate that PCSK9 deficiency in cultured hepatocytes increased the uptake and accumulation of saturated and unsaturated FAs. In the presence of saturated FAs, PCSK9 also protected cultured hepatocytes from ER stress and cytotoxicity. In line with these findings, a metabolic challenge using a high-fat diet caused severe hepatic steatosis, ER stress inflammation and fibrosis in the livers of Pcsk9 -/- mice compared to controls. Given that inhibition of CD36 ablated the observed accumulation of lipid in vitro and in vivo, our findings also highlight CD36 as a strong contributor to steatosis and liver injury in the context of PCSK9 deficiency. CONCLUSIONS: Collectively, our findings demonstrate that PCSK9 regulates hepatic triglyceride content in a manner dependent on CD36. In the presence of excess dietary fats, PCSK9 can also protect against hepatic steatosis and liver injury. LAY SUMMARY: The proprotein convertase subtilisin/kexin type 9 (PCSK9) is a circulating protein known to reduce the abundance of receptors on the surface of liver cells charged with the task of lipid uptake from the circulation. Although PCSK9 deficiency is known to cause lipid accumulation in mice and in cultured cells, the toxicological implications of this observation have not yet been reported. In this study, we demonstrate that PCSK9 can protect against cytotoxicity in cultured liver cells treated with a saturated fatty acid and we also show that Pcsk9 knockout mice develop increased liver injury in response to a high-fat diet.