Assessments of prostate cancer cell functions highlight differences between a pan-PI3K/mTOR inhibitor, gedatolisib, and single-node inhibitors of the PI3K/AKT/mTOR pathway.

Metastatic castration-resistant prostate cancer (mCRPC) is characterized by loss of androgen receptor (AR) sensitivity and oncogenic activation of the PI3K/AKT/mTOR (PAM) pathway. Loss of the PI3K regulator PTEN is frequent during prostate cancer (PC) initiation, progression, and therapeutic resistance. Co-targeting the PAM/AR pathways is a promising mCRPC treatment strategy but is hampered by reciprocal negative feedback inhibition or feedback relief. Most PAM inhibitors selectively spare (or weakly inhibit) one or more key nodes of the PAM pathway, potentiating drug resistance depending on the PAM pathway mutation status of patients. We posited that gedatolisib, a uniformly potent inhibitor of all class I PI3K isoforms, as well as mTORC1 and mTORC2, would be more effective than inhibitors targeting single PAM pathway nodes in PC cells. Using a combination of functional and metabolic assays, we evaluated a panel of PC cell lines with different PTEN/PIK3CA status for their sensitivity to multi-node PAM inhibitors (PI3K/mTOR: gedatolisib, samotolisib) and single-node PAM inhibitors (PI3Kα: alpelisib; AKT: capivasertib; mTOR: everolimus). Gedatolisib induced anti-proliferative and cytotoxic effects with greater potency and efficacy relative to the other PAM inhibitors, independent of PTEN/PIK3CA status. The superior effects of gedatolisib were likely associated with more effective inhibition of critical PAM-controlled cell functions, including cell cycle, survival, protein synthesis, oxygen consumption rate, and glycolysis. Our results indicate that potent and simultaneous blockade of all class I PI3K isoforms, mTORC1, and mTORC2 could circumvent PTEN-dependent resistance. Gedatolisib, as a single agent and in combination with other therapies, reported promising preliminary efficacy and safety in various solid tumor types. Gedatolisib is currently being evaluated in a Phase 1/2 clinical trial in combination with darolutamide in patients with mCRPC previously treated with an AR inhibitor, and in a Phase 3 clinical trial in combination with palbociclib and fulvestrant in patients with HR+/HER2- advanced breast cancer.

Gedatolisib shows superior potency and efficacy versus single-node PI3K/AKT/mTOR inhibitors in breast cancer models.

The PI3K, AKT, and mTOR (PAM) pathway is frequently dysregulated in breast cancer (BC) to accommodate high catabolic and anabolic activities driving tumor growth. Current therapeutic options for patients with hormone receptor (HR) + / HER2- advanced BC (ABC) include PAM inhibitors that selectively inhibit only one PAM pathway node, which can lead to drug resistance as cells rapidly adapt to maintain viability. We hypothesized that gedatolisib, which potently inhibits all Class I PI3K isoforms, as well as mTORC1 and mTORC2, may be more effective in BC cells than single-node PAM inhibitors by limiting adaptive resistances. By using multiple functional assays, a panel of BC cell lines was evaluated for their sensitivity to four different PAM inhibitors: gedatolisib (pan-PI3K/mTOR inhibitor), alpelisib (PI3Kα inhibitor), capivasertib (AKT inhibitor), and everolimus (mTORC1 inhibitor). Gedatolisib exhibited more potent and efficacious anti-proliferative and cytotoxic effects regardless of the PAM pathway mutational status of the cell lines compared to the single-node PAM inhibitors. The higher efficacy of gedatolisib was confirmed in three-dimensional culture and in BC PDX models. Mechanistically, gedatolisib decreased cell survival, DNA replication, cell migration and invasion, protein synthesis, glucose consumption, lactate production, and oxygen consumption more effectively than the other PAM inhibitors tested. These results indicate that inhibition of multiple PAM pathway nodes by a pan-PI3K/mTOR inhibitor like gedatolisib may be more effective at inducing anti-tumor activity than single-node PAM inhibitors. A global Phase 3 study is currently evaluating gedatolisib plus fulvestrant with and without palbociclib in patients with HR+/HER2- ABC.

Functional signaling test identifies HER2 negative breast cancer patients who may benefit from c-Met and pan-HER combination therapy.

BACKGROUND: Research is revealing the complex coordination between cell signaling systems as they adapt to genetic and epigenetic changes. Tools to uncover these highly complex functional linkages will play an important role in advancing more efficacious disease treatments. Current tumor cell signal transduction research is identifying coordination between receptor types, receptor families, and transduction pathways to maintain tumor cell viability despite challenging tumor microenvironment conditions. METHODS: In this report, coactivated abnormal levels of signaling activity for c-Met and HER family receptors in live tumor cells were measured by a new clinical test to identify a subpopulation of breast cancer patients that could be responsive to combined targeted therapies. The CELsignia Multi-Pathway Signaling Function (CELsignia) Test uses an impedance biosensor to quantify an individual patient's ex vivo live tumor cell signaling response in real-time to specific HER family and c-Met co-stimulation and targeted therapies. RESULTS: The test identified breast tumors with hyperactive HER1, HER2, HER3/4, and c-Met coordinated signaling that express otherwise normal amounts of these receptors. The supporting data of the pre-clinical verification of this test included analyses of 79 breast cancer patients' cell response to HER and c-Met agonists. The signaling results were confirmed using clinically approved matching targeted drugs, and combinations of targeted drugs in addition to correlative mouse xenograft tumor response to HER and c-Met targeted therapies. CONCLUSIONS: The results of this study demonstrated the potential benefit of a functional test for identifying a subpopulation of breast cancer patients with coordinated abnormal HER and c-Met signaling for a clinical trial testing combination targeted therapy. Video Abstract.

Lipid Droplet-Derived Monounsaturated Fatty Acids Traffic via PLIN5 to Allosterically Activate SIRT1.

Lipid droplets (LDs) provide a reservoir for triacylglycerol storage and are a central hub for fatty acid trafficking and signaling in cells. Lipolysis promotes mitochondrial biogenesis and oxidative metabolism via a SIRT1/PGC-1α/PPARα-dependent pathway through an unknown mechanism. Herein, we identify that monounsaturated fatty acids (MUFAs) allosterically activate SIRT1 toward select peptide-substrates such as PGC-1α. MUFAs enhance PGC-1α/PPARα signaling and promote oxidative metabolism in cells and animal models in a SIRT1-dependent manner. Moreover, we characterize the LD protein perilipin 5 (PLIN5), which is known to enhance mitochondrial biogenesis and function, to be a fatty-acid-binding protein that preferentially binds LD-derived monounsaturated fatty acids and traffics them to the nucleus following cAMP/PKA-mediated lipolytic stimulation. Thus, these studies identify the first-known endogenous allosteric modulators of SIRT1 and characterize a LD-nuclear signaling axis that underlies the known metabolic benefits of MUFAs and PLIN5.

Integrated Regulation of Hepatic Lipid and Glucose Metabolism by Adipose Triacylglycerol Lipase and FoxO Proteins.

Metabolism is a highly integrated process that is coordinately regulated between tissues and within individual cells. FoxO proteins are major targets of insulin action and contribute to the regulation of gluconeogenesis, glycolysis, and lipogenesis in the liver. However, the mechanisms by which FoxO proteins exert these diverse effects in an integrated fashion remain poorly understood. We report that FoxO proteins also exert important effects on intrahepatic lipolysis and fatty acid oxidation via the regulation of adipose triacylglycerol lipase (ATGL), which mediates the first step in lipolysis, and its inhibitor, the G0/S1 switch 2 gene (G0S2). We also find that ATGL-dependent lipolysis plays a critical role in mediating diverse effects of FoxO proteins in the liver, including effects on gluconeogenic, glycolytic, and lipogenic gene expression and metabolism. These results indicate that intrahepatic lipolysis plays a critical role in mediating and integrating the regulation of glucose and lipid metabolism downstream of FoxO proteins.

Quantitative analysis of the murine lipid droplet-associated proteome during diet-induced hepatic steatosis.

Hepatic steatosis is characterized by the accumulation of lipid droplets (LDs), which are composed of a neutral lipid core surrounded by a phospholipid monolayer embedded with many proteins. Although the LD-associated proteome has been investigated in multiple tissues and organisms, the dynamic changes in the murine LD-associated proteome in response to obesity and hepatic steatosis have not been studied. We characterized the hepatic LD-associated proteome of C57BL/6J male mouse livers following high-fat feeding using isobaric tagging for relative and absolute quantification. Of the 1,520 proteins identified with a 5% local false discovery rate, we report a total of 48 proteins that were increased and 52 proteins that were decreased on LDs in response to high-fat feeding. Most notably, ribosomal and endoplasmic reticulum proteins were increased and extracellular and cytosolic proteins were decreased in response to high-fat feeding. Additionally, many proteins involved in fatty acid catabolism or xenobiotic metabolism were enriched in the LD fraction following high-fat feeding. In contrast, proteins involved in glucose metabolism and liver X receptor or retinoid X receptor activation were decreased on LDs of high-fat-fed mice. This study provides insights into unique biological functions of hepatic LDs under normal and steatotic conditions.

Hepatic lipid droplet biology: Getting to the root of fatty liver.

Hepatic steatosis is defined by the accumulation of lipid droplets (LDs). Once thought to be only inert energy storage depots, LDs are increasingly recognized as organelles that have important functions in hepatocytes beyond lipid storage. The lipid and protein composition of LDs is highly dynamic and influences their intrinsic metabolism and signaling properties, which ultimately links them to the changes in hepatic function. This concise review highlights recent discoveries in LD biology and unique aspects of hepatic LDs and their role in liver disease.

ATGL-catalyzed lipolysis regulates SIRT1 to control PGC-1α/PPAR-α signaling.

Sirtuin 1 (SIRT1), an NAD(+)-dependent protein deacetylase, regulates a host of target proteins, including peroxisome proliferator-activated receptor (PPAR)-γ coactivator-1α (PGC-1α), a transcriptional coregulator that binds to numerous transcription factors in response to deacetylation to promote mitochondrial biogenesis and oxidative metabolism. Our laboratory and others have shown that adipose triglyceride lipase (ATGL) increases the activity of the nuclear receptor PPAR-α, a PGC-1α binding partner, to promote fatty acid oxidation. Fatty acids bind and activate PPAR-α; therefore, it has been presumed that fatty acids derived from ATGL-catalyzed lipolysis act as PPAR-α ligands. We provide an alternate mechanism that links ATGL to PPAR-α signaling. We show that SIRT1 deacetylase activity is positively regulated by ATGL to promote PGC-1α signaling. In addition, ATGL mediates the effects of ß-adrenergic signaling on SIRT1 activity, and PGC-1α and PPAR-α target gene expression independent of changes in NAD(+). Moreover, SIRT1 is required for the induction of PGC-1α/PPAR-α target genes and oxidative metabolism in response to increased ATGL-mediated lipolysis. Taken together, this work identifies SIRT1 as a critical node that links ß-adrenergic signaling and lipolysis to changes in the transcriptional regulation of oxidative metabolism.

Glucose metabolism in mammalian cell culture: new insights for tweaking vintage pathways.

Cultured mammalian cells are major vehicles for producing therapeutic proteins, and energy metabolism in those cells profoundly affects process productivity. The characteristic high glucose consumption and lactate production of industrial cell lines as well as their adverse effects on productivity have been the target of both cell line and process improvement for several decades. Recent research advances have shed new light on regulation of glucose metabolism and its links to cell proliferation. This review highlights our current understanding in this area of crucial importance in bioprocessing and further discusses strategies for harnessing new findings toward process enhancement through the manipulation of cellular energy metabolism.

Inhibition of glucokinase translocation by AMP-activated protein kinase is associated with phosphorylation of both GKRP and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

The rate of glucose phosphorylation in hepatocytes is determined by the subcellular location of glucokinase and by its association with its regulatory protein (GKRP) in the nucleus. Elevated glucose concentrations and precursors of fructose 1-phosphate (e.g., sorbitol) cause dissociation of glucokinase from GKRP and translocation to the cytoplasm. In this study, we investigated the counter-regulation of substrate-induced translocation by AICAR (5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside), which is metabolized by hepatocytes to an AMP analog, and causes activation of AMP-activated protein kinase (AMPK) and depletion of ATP. During incubation of hepatocytes with 25 mM glucose, AICAR concentrations below 200 microM activated AMPK without depleting ATP and inhibited glucose phosphorylation and glucokinase translocation with half-maximal effect at 100-140 microM. Glucose phosphorylation and glucokinase translocation correlated inversely with AMPK activity. AICAR also counteracted translocation induced by a glucokinase activator and partially counteracted translocation by sorbitol. However, AICAR did not block the reversal of translocation (from cytoplasm to nucleus) after substrate withdrawal. Inhibition of glucose-induced translocation by AICAR was greater than inhibition by glucagon and was associated with phosphorylation of both GKRP and the cytoplasmic glucokinase binding protein, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2) on ser-32. Expression of a kinase-active PFK2 variant lacking ser-32 partially reversed the inhibition of translocation by AICAR. Phosphorylation of GKRP by AMPK partially counteracted its inhibitory effect on glucokinase activity, suggesting altered interaction of glucokinase and GKRP. In summary, mechanisms downstream of AMPK activation, involving phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and GKRP are involved in the ATP-independent inhibition of glucose-induced glucokinase translocation by AICAR in hepatocytes.

A role for PFK-2/FBPase-2, as distinct from fructose 2,6-bisphosphate, in regulation of insulin secretion in pancreatic beta-cells.

PFK-2/FBPase-2 (6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase) catalyses the formation and degradation of fructose 2,6-P(2) (fructose 2,6-bisphosphate) and is also a glucokinase-binding protein. The role of fructose 2,6-P(2) in regulating glucose metabolism and insulin secretion in pancreatic beta-cells is unresolved. We down-regulated the endogenous isoforms of PFK-2/FBPase-2 with siRNA (small interfering RNA) and expressed KA (kinase active) and KD (kinase deficient) variants to distinguish between the role of PFK-2/FBPase-2 protein and the role of its product, fructose 2,6-P(2), in regulating beta-cell function. Human islets expressed the PFKFB2 (the gene encoding isoform 2 of the PFK2/FBPase2 protein) and PFKFB3 (the gene encoding isoform 3 of the PFK2/FBPase2 protein) isoforms and mouse islets expressed PFKFB2 at the mRNA level [RT-PCR (reverse transcription-PCR)]. Rat islets expressed PFKFB2 lacking the C-terminal phosphorylation sites. The glucose-responsive MIN6 and INS1E cell lines expressed PFKFB2 and PFKFB3. PFK-2 activity and the cell content of fructose 2,6-P(2) were increased by elevated glucose concentration and during pharmacological activation of AMPK (AMP-activated protein kinase), which also increased insulin secretion. Partial down-regulation of endogenous PFKFB2 and PFKFB3 in INS1E by siRNA decreased PFK-2/FBPase-2 protein, fructose 2,6-P(2) content, glucokinase activity and glucoseinduced insulin secretion. Selective down-regulation of glucose-induced fructose 2,6-P(2) in the absence of down-regulation of PFK-2/FBPase-2 protein, using a KD PFK-2/FBPase-2 variant, resulted in sustained glycolysis and elevated glucose-induced insulin secretion, indicating an over-riding role of PFK-2/FBPase-2 protein, as distinct from its product fructose 2,6-P(2), in potentiating glucose-induced insulin secretion. Whereas down-regulation of PFK-2/FBPase-2 decreased glucokinase activity, overexpression of PFK-2/FBPase-2 only affected glucokinase distribution. It is concluded that PFK-2/FBPase-2 protein rather than its product fructose 2,6-P(2) is the over-riding determinant of glucose-induced insulin secretion through regulation of glucokinase activity or subcellular targeting.

Roles for fructose-2,6-bisphosphate in the control of fuel metabolism: beyond its allosteric effects on glycolytic and gluconeogenic enzymes.

Fructose-2,6-bisphosphate (F26P2) was identified as a regulator of glucose metabolism over 25 years ago. A truly bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PFK2/FBP2), with two active sites synthesizes F26P2 from fructose-6-phosphate (F6P) and ATP or degrades F26P2 to F6P and Pi. In the classic view, F26P2 regulates glucose metabolism by allosteric effects on 6-phosphofructo-1-kinase (6PFK1, activation) and fructose-1,6-bisphosphatase (FBPase, inhibition). When levels of F26P2 are high, glycolysis is enhanced and gluconeogenesis is inhibited. In this regard, altering levels of F26P2 via 6PFK2/FBP2 overexpression has been used for metabolic modulation, and has been shown capable of restoring euglycemia in rodent models of diabetes. Recently, a number of novel observations have suggested that F26P2 has much broader effects on the enzymes of glucose metabolism. This is evidenced by the effects of F26P2 on the gene expression of two key glucose metabolic enzymes, glucokinase (GK) and glucose-6-phosphatase (G6Pase). When levels of F26P2 are elevated in the liver, the gene expression and protein amount of GK is increased whereas G6Pase is decreased. These coordinated changes in GK and G6Pase protein illustrate how F26P2 regulates glucose metabolism. F26P2 also affects the gene expression of enzymes related to lipid metabolism. When F26P2 levels are elevated in liver, the expression of two key lipogenic enzymes, acetyl-CoA carboxylase 1 (ACC1) and fatty acid synthase (FAS) is reduced, contributing to a unique coordinated decrease in lipogenesis. When combined, F26P2 effects on glucose and lipid metabolism provide cooperative regulation of fuel metabolism. The regulatory roles for F26P2 have also expanded to transcription factors, as well as certain key proteins (enzymes) of signaling and/or energy sensoring. Although some effects may be secondary to changes in metabolite levels, high levels of F26P2 have been shown to regulate protein amount and/or phosphorylation state of hepatic nuclear factor 1-alpha (HNF1alpha), carbohydrate response element binding protein (ChREBP), peroxisome proliferators-activated receptor alpha (PPARalpha), and peroxisome proliferators-activated receptor gamma co-activator 1beta (PGC1beta), as well as Akt and AMP-activated protein kinase (AMPK). Importantly, changes in these transcription factors, signaling proteins, and sensor proteins are produced in a way that appropriately coordinates whole body fuel metabolism.

Perturbation of glucose flux in the liver by decreasing F26P2 levels causes hepatic insulin resistance and hyperglycemia.

Hepatic insulin resistance is one of the characteristics of type 2 diabetes and contributes to the development of hyperglycemia. How changes in hepatic glucose flux lead to insulin resistance is not clearly defined. We determined the effects of decreasing the levels of hepatic fructose 2,6-bisphosphate (F26P(2)), a key regulator of glucose metabolism, on hepatic glucose flux in the normal 129J mice. Upon adenoviral overexpression of a kinase activity-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, the enzyme that determines F26P(2) level, hepatic F26P(2) levels were decreased twofold compared with those of control virus-treated mice in basal state. In addition, under hyperinsulinemic conditions, hepatic F26P(2) levels were much lower than those of the control. The decrease in F26P(2) leads to the elevation of basal and insulin-suppressed hepatic glucose production. Also, the efficiency of insulin to suppress hepatic glucose production was decreased (63.3 vs. 95.5% suppression of the control). At the molecular level, a decrease in insulin-stimulated Akt phosphorylation was consistent with hepatic insulin resistance. In the low hepatic F26P(2) states, increases in both gluconeogenesis and glycogenolysis in the liver are responsible for elevations of hepatic glucose production and thereby contribute to the development of hyperglycemia. Additionally, the increased hepatic gluconeogenesis was associated with the elevated mRNA levels of peroxisome proliferator-activated receptor-gamma coactivator-1alpha and phosphoenolpyruvate carboxykinase. This study provides the first in vivo demonstration showing that decreasing hepatic F26P(2) levels leads to increased gluconeogenesis in the liver. Taken together, the present study demonstrates that perturbation of glucose flux in the liver plays a predominant role in the development of a diabetic phenotype, as characterized by hepatic insulin resistance.

Enhancing hepatic glycolysis reduces obesity: differential effects on lipogenesis depend on site of glycolytic modulation.

Reducing obesity requires an elevation of energy expenditure and/or a suppression of food intake. Here we show that enhancing hepatic glycolysis reduces body weight and adiposity in obese mice. Overexpression of glucokinase or 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is used to increase hepatic glycolysis. Either of the two treatments produces similar increases in rates of fatty acid oxidation in extrahepatic tissues, i.e., skeletal muscle, leading to an elevation of energy expenditure. However, only 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase overexpression causes a suppression of food intake and a decrease in hypothalamic neuropeptide Y expression, contributing to a more pronounced reduction of body weight with this treatment. Furthermore, the two treatments cause differential lipid profiles due to opposite effects on hepatic lipogenesis, associated with distinct phosphorylation states of carbohydrate response element binding protein and AMP-activated protein kinase. The step at which hepatic glycolysis is enhanced dramatically influences overall whole-body energy balance and lipid profiles.