A strategy for dual inhibition of the proteasome and fatty acid synthase with belactosin C-orlistat hybrids.

The proteasome, a validated cellular target for cancer, is central for maintaining cellular homeostasis, while fatty acid synthase (FAS), a novel target for numerous cancers, is responsible for palmitic acid biosynthesis. Perturbation of either enzymatic machine results in decreased proliferation and ultimately cellular apoptosis. Based on structural similarities, we hypothesized that hybrid molecules of belactosin C, a known proteasome inhibitor, and orlistat, a known inhibitor of the thioesterase domain of FAS, could inhibit both enzymes. Herein, we describe proof-of-principle studies leading to the design, synthesis and enzymatic activity of several novel, ß-lactone-based, dual inhibitors of these two enzymes. Validation of dual enzyme targeting through activity-based proteome profiling with an alkyne probe modeled after the most potent inhibitor, and preliminary serum stability studies of selected derivatives are also described. These results provide proof of concept for dual targeting of the proteasome and fatty acid synthase-thioesterase (FAS-TE) enabling a new approach for the development of drug-candidates with potential to overcome resistance.

Inactivation of Adenomatous Polyposis Coli Reduces Bile Acid/Farnesoid X Receptor Expression through Fxr gene CpG Methylation in Mouse Colon Tumors and Human Colon Cancer Cells.

BACKGROUND: The farnesoid X receptor (FXR) regulates bile acid (BA) metabolism and possesses tumor suppressor functions. FXR expression is reduced in colorectal tumors of subjects carrying inactivated adenomatous polyposis coli (APC). Identifying the mechanisms responsible for this reduction may offer new molecular targets for colon cancer prevention. OBJECTIVE: We investigated how APC inactivation influences the regulation of FXR expression in colonic mucosal cells. We hypothesized that APC inactivation would epigenetically repress nuclear receptor subfamily 1, group H, member 4 (FXR gene name) expression through increased CpG methylation. METHODS: Normal proximal colonic mucosa and normal-appearing adjacent colonic mucosa and colon tumors were collected from wild-type C57BL/6J and Apc-deficient (Apc(Min) (/+)) male mice, respectively. The expression of Fxr, ileal bile acid-binding protein (Ibabp), small heterodimer partner (Shp), and cyclooxygenase-2 (Cox-2) were determined by real-time polymerase chain reaction. In both normal and adjacent colonic mucosa and colon tumors, we measured CpG methylation of Fxr in bisulfonated genomic DNA. In vitro, we measured the impact of APC inactivation and deoxycholic acid (DCA) treatment on FXR expression in human colon cancer HCT-116 cells transfected with silencing RNA for APC and HT-29 cells carrying inactivated APC. RESULTS: In Apc(Min) (/+) mice, constitutive CpG methylation of the Fxrα3/4 promoter was linked to reduced (60-90%) baseline Fxr, Ibabp, and Shp and increased Cox-2 expression in apparently normal adjacent mucosa and colon tumors. Apc knockdown in HCT-116 cells increased cellular myelocytomatosis (c-MYC) and lowered (∼50%) FXR expression, which was further reduced (∼80%) by DCA. In human HCT-116 but not HT-29 colon cancer cells, DCA induced FXR expression and lowered CpG methylation of FXR. CONCLUSIONS: We conclude that the loss of APC function favors the silencing of FXR expression through CpG hypermethylation in mouse colonic mucosa and human colon cells, leading to reduced expression of downstream targets (SHP, IBABP) involved in BA homeostasis while increasing the expression of factors (COX-2, c-MYC) that contribute to inflammation and colon cancer.

Right on TARGET: glutamine metabolism in cancer.

Recent studies highlight the importance of glutamine metabolism in metabolic reprogramming, which underlies cancer cell addiction to glutamine. Examples for the dependence on glutamine metabolism are seen across different tumor types as during different phases of cancer development, progression and response to therapy. In this perspective, we assess the possibility of targeting glutamine metabolism as a therapeutic modality for cancer.

High-Throughput Multiplexed Peptide-Centric Profiling Illustrates Both Substrate Cleavage Redundancy and Specificity in the MMP Family.

Matrix metalloproteinases (MMPs) play incompletely understood roles in health and disease. Knowing the MMP cleavage preferences is essential for a better understanding of the MMP functions and design of selective inhibitors. To elucidate the cleavage preferences of MMPs, we employed a high-throughput multiplexed peptide-centric profiling technology involving the cleavage of 18,583 peptides by 18 proteinases from the main sub-groups of the MMP family. Our results enabled comparison of the MMP substrates on a global scale, leading to the most efficient and selective substrates. The data validated the accuracy of our cleavage prediction software. This software allows us and others to locate, with nearly 100% accuracy, the MMP cleavage sites in the peptide sequences. In addition to increasing our understanding of both the selectivity and the redundancy of the MMP family, our study generated a roadmap for the subsequent MMP structural-functional studies and efficient substrate and inhibitor design.

Glutamate and asparagine cataplerosis underlie glutamine addiction in melanoma.

Glutamine dependence is a prominent feature of cancer metabolism, and here we show that melanoma cells, irrespective of their oncogenic background, depend on glutamine for growth. A quantitative audit of how carbon from glutamine is used showed that TCA-cycle-derived glutamate is, in most melanoma cells, the major glutamine-derived cataplerotic output and product of glutaminolysis. In the absence of glutamine, TCA cycle metabolites were liable to depletion through aminotransferase-mediated α-ketoglutarate-to-glutamate conversion and glutamate secretion. Aspartate was an essential cataplerotic output, as melanoma cells demonstrated a limited capacity to salvage external aspartate. Also, the absence of asparagine increased the glutamine requirement, pointing to vulnerability in the aspartate-asparagine biosynthetic pathway within melanoma metabolism. In contrast to melanoma cells, melanocytes could grow in the absence of glutamine. Melanocytes use more glutamine for protein synthesis rather than secreting it as glutamate and are less prone to loss of glutamate and TCA cycle metabolites when starved of glutamine.

Regulation of glutamine carrier proteins by RNF5 determines breast cancer response to ER stress-inducing chemotherapies.

Many tumor cells are fueled by altered metabolism and increased glutamine (Gln) dependence. We identify regulation of the L-glutamine carrier proteins SLC1A5 and SLC38A2 (SLC1A5/38A2) by the ubiquitin ligase RNF5. Paclitaxel-induced ER stress to breast cancer (BCa) cells promotes RNF5 association, ubiquitination, and degradation of SLC1A5/38A2. This decreases Gln uptake, levels of TCA cycle components, mTOR signaling, and proliferation while increasing autophagy and cell death. Rnf5-deficient MMTV-PyMT mammary tumors were less differentiated and showed elevated SLC1A5 expression. Whereas RNF5 depletion in MDA-MB-231 cells promoted tumorigenesis and abolished paclitaxel responsiveness, SLC1A5/38A2 knockdown elicited opposing effects. Inverse RNF5(hi)/SLC1A5/38A2(lo) expression was associated with positive prognosis in BCa. Thus, RNF5 control of Gln uptake underlies BCa response to chemotherapies.

4EBP1/eIF4E and p70S6K/RPS6 axes play critical and distinct roles in hepatocarcinogenesis driven by AKT and N-Ras proto-oncogenes in mice.

UNLABELLED: Concomitant expression of activated forms of v-akt murine thymoma viral oncogene homolog (AKT) and Ras in mouse liver (AKT/Ras) leads to rapid tumor development through strong activation of the mammalian target of rapamycin complex 1 (mTORC1) pathway. mTORC1 functions by regulating p70S6K/ribosomal protein S6 (RPS6) and eukaryotic translation initiation factor 4E-binding protein 1/ eukaryotic translation initiation factor 4E (4EBP1/eIF4E) cascades. How these cascades contribute to hepatocarcinogenesis remains unknown. Here, we show that inhibition of the RPS6 pathway by rapamycin effectively suppressed, whereas blockade of the 4EBP1/eIF4E cascade by 4EBP1A4, an unphosphorylatable form of 4EBP1, significantly delayed, AKT/Ras-induced hepatocarcinogenesis. Combined treatment with rapamycin and 4EBP1A4 completely inhibited AKT/Ras hepatocarcinogenesis. This strong antineoplastic effect was successfully recapitulated by ablating regulatory associated protein of mTORC1, the major subunit of mTORC1, in AKT/Ras-overexpressing livers. Furthermore, we demonstrate that overexpression of eIF4E, the proto-oncogene whose activity is specifically inhibited by 4EBP1, resulted in hepatocellular carcinoma (HCC) development in cooperation with activated Ras. Mechanistically, we identified the ectonucleoside triphosphate diphosphohydrolase 5/ adenylate kinase 1/cytidine monophosphate kinase 1 axis and the mitochondrial biogenesis pathway as targets of the 4EBP1/eIF4E cascade in AKT/Ras and Ras/eIF4E livers as well as in human HCC cell lines and tissues. CONCLUSIONS: Complete inhibition of mTORC1 is required to suppress liver cancer development induced by AKT and Ras proto-oncogenes in mice. The mTORC1 effectors, RPS6 and eIF4E, play distinct roles and are both necessary for AKT/Ras hepatocarcinogenesis. These new findings might open the way for innovative therapies against human HCC.

Basis for substrate recognition and distinction by matrix metalloproteinases.

Genomic sequencing and structural genomics produced a vast amount of sequence and structural data, creating an opportunity for structure-function analysis in silico [Radivojac P, et al. (2013) Nat Methods 10(3):221-227]. Unfortunately, only a few large experimental datasets exist to serve as benchmarks for function-related predictions. Furthermore, currently there are no reliable means to predict the extent of functional similarity among proteins. Here, we quantify structure-function relationships among three phylogenetic branches of the matrix metalloproteinase (MMP) family by comparing their cleavage efficiencies toward an extended set of phage peptide substrates that were selected from ∼ 64 million peptide sequences (i.e., a large unbiased representation of substrate space). The observed second-order rate constants [k(obs)] across the substrate space provide a distance measure of functional similarity among the MMPs. These functional distances directly correlate with MMP phylogenetic distance. There is also a remarkable and near-perfect correlation between the MMP substrate preference and sequence identity of 50-57 discontinuous residues surrounding the catalytic groove. We conclude that these residues represent the specificity-determining positions (SDPs) that allowed for the expansion of MMP proteolytic function during evolution. A transmutation of only a few selected SDPs proximal to the bound substrate peptide, and contributing the most to selectivity among the MMPs, is sufficient to enact a global change in the substrate preference of one MMP to that of another, indicating the potential for the rational and focused redesign of cleavage specificity in MMPs.

Functional specialization in proline biosynthesis of melanoma.

Proline metabolism is linked to hyperprolinemia, schizophrenia, cutis laxa, and cancer. In the latter case, tumor cells tend to rely on proline biosynthesis rather than salvage. Proline is synthesized from either glutamate or ornithine; both are converted to pyrroline-5-carboxylate (P5C), and then to proline via pyrroline-5-carboxylate reductases (PYCRs). Here, the role of three isozymic versions of PYCR was addressed in human melanoma cells by tracking the fate of (13)C-labeled precursors. Based on these studies we conclude that PYCR1 and PYCR2, which are localized in the mitochondria, are primarily involved in conversion of glutamate to proline. PYCRL, localized in the cytosol, is exclusively linked to the conversion of ornithine to proline. This analysis provides the first clarification of the role of PYCRs to proline biosynthesis.

Glutamine-fueled mitochondrial metabolism is decoupled from glycolysis in melanoma.

In this perspective, we revise the historic notion that cancer is a disease of mitochondria. We summarize recent findings on the function and rewiring of central carbon metabolism in melanoma. Metabolic profiling studies using stable isotope tracers show that glycolysis is decoupled from the tricarboxylic acid (TCA) cycle. This decoupling is not 'dysfunction' but rather an alternate wiring required by tumor cells to remain metabolically versatile. In large part, this requirement is met by glutamine feeding the TCA cycle as an alternative source of carbon. Glutamine is also used in non-conventional ways, like traveling in reverse through the TCA flux to feed fatty acid biosynthesis. Biosynthetic networks linked with non-essential amino acids alanine, serine, arginine, and proline are also significantly impacted by the use of glutamine as an alternate carbon source.

Reverse TCA cycle flux through isocitrate dehydrogenases 1 and 2 is required for lipogenesis in hypoxic melanoma cells.

The tricarboxylic acid (TCA) cycle is the central hub of oxidative metabolism, running in the classic forward direction to provide carbon for biosynthesis and reducing agents for generation of ATP. Our metabolic tracer studies in melanoma cells showed that in hypoxic conditions the TCA cycle is largely disconnected from glycolysis. By studying the TCA branch point metabolites, acetyl CoA and citrate, as well as the metabolic endpoint glutamine and fatty acids, we developed a comprehensive picture of the rewiring of the TCA cycle that occurs in hypoxia. Hypoxic tumor cells maintain proliferation by running the TCA cycle in reverse. The source of carbon for acetyl CoA, citrate, and fatty acids switches from glucose in normoxia to glutamine in hypoxia. This hypoxic flux from glutamine into fatty acids is mediated by reductive carboxylation. This reductive carboxylation is catalyzed by two isocitrate dehydrogenases, IDH1 and IDH2. Their combined action is necessary and sufficient to effect the reverse TCA flux and maintain cellular viability.

Unusual binding of ursodeoxycholic acid to ileal bile acid binding protein: role in activation of FXRα.

Ursodeoxycholic acid (UDCA, ursodiol) is used to prevent damage to the liver in patients with primary biliary cirrhosis. The drug also prevents the progression of colorectal cancer and the recurrence of high-grade colonic dysplasia. However, the molecular mechanism by which UDCA elicits its beneficial effects is not entirely understood. The aim of this study was to determine whether ileal bile acid binding protein (IBABP) has a role in mediating the effects of UDCA. We find that UDCA binds to a single site on IBABP and increases the affinity for major human bile acids at a second binding site. As UDCA occupies one of the bile acid binding sites on IBABP, it reduces the cooperative binding that is often observed for the major human bile acids. Furthermore, IBABP is necessary for the full activation of farnesoid X receptor α (FXRα) by bile acids, including UDCA. These observations suggest that IBABP may have a role in mediating some of the intestinal effects of UDCA.

Comparative proteomic analysis of a cytosolic fraction from ß3 integrin-deficient cells.

Integrins are heterodimeric transmembrane receptors involved in sensing and transmitting informational cues from the extracellular environment to the cell. This study explored sub-proteome changes in response to elimination of the ß3 integrin using a knockout murine model. Cleavable isotope-coded affinity tagging (cICAT) in combination with sub-cellular fractionation, multiple dimensions of separation and tandem mass spectrometry (MS/MS) were used to characterize differentially expressed proteins among ß3 integrin(-/-) (ß3(-/-)) mouse embryonic fibroblasts and isogenic wild-type (WT) controls. From a cytosolic protein fraction, 48 proteins were identified, in which expression differed by > 1.5-fold. Predominant ontological groups included actin-binding/cytoskeletal proteins and protease/protease inhibitors. Interestingly, ß3 integrin expression was inversely correlated with expression of cathepsin B, a lysosomal cysteine protease, as its expression was greater by over 3.5-fold in the ß3(-/-) cells. This inverse correlation was also observed in stable heterologous cells transfected with ß3 integrin, where the intracellular expression and activity of cathepsin B was lower compared to control cells. Our data suggests that the composition of the cellular proteome is influenced by integrin expression patterns and reveals a strong functional relationship between ß3 integrin and cathepsin B.

Risk modification of colorectal adenoma by CYP7A1 polymorphisms and the role of bile acid metabolism in carcinogenesis.

Cholesterol 7α-hydroxylase (CYP7A1), the rate-limiting enzyme in the conversion of cholesterol to bile acids, is a postulated gene modifier of colorectal cancer risk and target for the therapeutic bile acid, ursodeoxycholic acid (UDCA). We investigated associations between CYP7A1 polymorphisms and fecal bile acids, colorectal adenoma (CRA), and UDCA efficacy for CRA prevention. Seven tagging, single-nucleotide polymorphisms (SNP) in CYP7A1 were measured in 703 (355 UDCA, 348 placebo) participants of a phase III chemoprevention trial, of which 495 had known baseline fecal bile acid concentrations. In the placebo arm, participants with two minor G(rs8192871) alleles (tag for a low activity promoter polymorphism at -204) had lower odds of high secondary bile acids (OR = 0.26, 95% CI: 0.10-0.69), and CRA at 3 years' follow-up (OR = 0.41, 95% CI: 0.19-0.89), than AA carriers. Haplotype construction from the six polymorphic SNPs showed participants with the third most common haplotype (C(rs10957057)C(rs8192879)G(rs8192877)T(rs11786580)A(rs8192871)G(rs13251096)) had higher odds of high primary bile acids (OR = 2.34, 95% CI: 1.12-4.89) and CRA (OR = 1.89, 95% CI: 1.00-3.57) than those with the most common CTACAG haplotype. Furthermore, three SNPs (rs8192877, rs8192871, and rs13251096) each modified UDCA efficacy for CRA prevention, and CCGTAG-haplotype carriers experienced 71% lower odds of CRA recurrence with UDCA treatment, an effect not present for other haplotypes (test for UDCA-haplotype interaction, P = 0.020). Our findings support CYP7A1 polymorphisms as determinants of fecal bile acids and risk factors for CRA. Furthermore, UDCA efficacy for CRA prevention may be modified by genetic variation in CYP7A1, limiting treatment benefit to a subgroup of the population.

Comparative metabolic flux profiling of melanoma cell lines: beyond the Warburg effect.

Metabolic rewiring is an established hallmark of cancer, but the details of this rewiring at a systems level are not well characterized. Here we acquire this insight in a melanoma cell line panel by tracking metabolic flux using isotopically labeled nutrients. Metabolic profiling and flux balance analysis were used to compare normal melanocytes to melanoma cell lines in both normoxic and hypoxic conditions. All melanoma cells exhibited the Warburg phenomenon; they used more glucose and produced more lactate than melanocytes. Other changes were observed in melanoma cells that are not described by the Warburg phenomenon. Hypoxic conditions increased fermentation of glucose to lactate in both melanocytes and melanoma cells (the Pasteur effect). However, metabolism was not strictly glycolytic, as the tricarboxylic acid (TCA) cycle was functional in all melanoma lines, even under hypoxia. Furthermore, glutamine was also a key nutrient providing a substantial anaplerotic contribution to the TCA cycle. In the WM35 melanoma line glutamine was metabolized in the "reverse" (reductive) direction in the TCA cycle, particularly under hypoxia. This reverse flux allowed the melanoma cells to synthesize fatty acids from glutamine while glucose was primarily converted to lactate. Altogether, this study, which is the first comprehensive comparative analysis of metabolism in melanoma cells, provides a foundation for targeting metabolism for therapeutic benefit in melanoma.

Proteins and an inflammatory network expressed in colon tumors.

The adenomatous polyposis coli (APC) protein is crucial to homeostasis of normal intestinal epithelia because it suppresses the ß-catenin/TCF pathway. Consequently, loss or mutation of the APC gene causes colorectal tumors in humans and mice. Here, we describe our use of multidimensional protein identification technology (MudPIT) to compare protein expression in colon tumors to that of adjacent healthy colon tissue from Apc(Min/+) mice. Twenty-seven proteins were found to be up-regulated in colon tumors and 25 were down-regulated. As an extension of the proteomic analysis, the differentially expressed proteins were used as "seeds" to search for coexpressed genes. This approach revealed a coexpression network of 45 genes that is up-regulated in colon tumors. Members of the network include the antibacterial peptide cathelicidin (CAMP), Toll-like receptors (TLRs), IL-8, and triggering receptor expressed on myeloid cells 1 (TREM1). The coexpression network is associated with innate immunity and inflammation, and there is significant concordance between its connectivity in humans versus mice (Friedman: p value = 0.0056). This study provides new insights into the proteins and networks that are likely to drive the onset and progression of colon cancer.

Polymer particle shape independently influences binding and internalization by macrophages.

The interaction of macrophages with micro and nanoparticles (MNPs) is important because these cells clear particles from the circulation, and because they are potential therapeutic targets in inflammatory conditions, atherosclerosis and cancer. Therefore, an understanding of the features of MNPs that influence their interaction with macrophages may allow optimization of their properties for enhanced drug delivery. In this study, we show that particle shape impacts phagocytosis by macrophages, and more importantly, that particle shape and size separately impact attachment and internalization. The study provides a methodology for further exploring how particle shape can be controlled to achieve desired attachment and internalization. The results of the study also give mechanistic guidance on how particle shape can be manipulated to design drug carriers to evade macrophages, or alternatively to target macrophages.

Mitochondrial p32 protein is a critical regulator of tumor metabolism via maintenance of oxidative phosphorylation.

p32/gC1qR/C1QBP/HABP1 is a mitochondrial/cell surface protein overexpressed in certain cancer cells. Here we show that knocking down p32 expression in human cancer cells strongly shifts their metabolism from oxidative phosphorylation (OXPHOS) to glycolysis. The p32 knockdown cells exhibited reduced synthesis of the mitochondrial-DNA-encoded OXPHOS polypeptides and were less tumorigenic in vivo. Expression of exogenous p32 in the knockdown cells restored the wild-type cellular phenotype and tumorigenicity. Increased glucose consumption and lactate production, known as the Warburg effect, are almost universal hallmarks of solid tumors and are thought to favor tumor growth. However, here we show that a protein regularly overexpressed in some cancers is capable of promoting OXPHOS. Our results indicate that high levels of glycolysis, in the absence of adequate OXPHOS, may not be as beneficial for tumor growth as generally thought and suggest that tumor cells use p32 to regulate the balance between OXPHOS and glycolysis.

Matrix metalloproteinase proteolysis of the myelin basic protein isoforms is a source of immunogenic peptides in autoimmune multiple sclerosis.

BACKGROUND: Matrix metalloproteinases (MMPs) play a significant role in the fragmentation of myelin basic protein (MBP) and demyelination leading to autoimmune multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). The classic MBP isoforms are predominantly expressed in the oligodendrocytes of the CNS. The splice variants of the single MBP gene (Golli-MBP BG21 and J37) are widely expressed in the neurons and also in the immune cells. The relative contribution of the individual MMPs to the MBP cleavage is not known. METHODOLOGY/PRINCIPAL FINDINGS: To elucidate which MMP plays the primary role in cleaving MBP, we determined the efficiency of MMP-2, MMP-8, MMP-9, MMP-10, MMP-12, MT1-MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP and MT6-MMP in the cleavage of the MBP, BG21 and J37 isoforms in the in vitro cleavage reactions followed by mass-spectroscopy analysis of the cleavage fragments. As a result, we identified the MMP cleavage sites and the sequence of the resulting fragments. We determined that MBP, BG21 and J37 are highly sensitive to redundant MMP proteolysis. MT6-MMP (initially called leukolysin), however, was superior over all of the other MMPs in cleaving the MBP isoforms. Using the mixed lymphocyte culture assay, we demonstrated that MT6-MMP proteolysis of the MBP isoforms readily generated, with a near quantitative yield, the immunogenic N-terminal 1-15 MBP peptide. This peptide selectively stimulated the proliferation of the PGPR7.5 T cell clone isolated from mice with EAE and specific for the 1-15 MBP fragment presented in the MHC H-2(U) context. CONCLUSIONS/SIGNIFICANCE: In sum, our biochemical observations led us to hypothesize that MT6-MMP, which is activated by furin and associated with the lipid rafts, plays an important role in MS pathology and that MT6-MMP is a novel and promising drug target in MS especially when compared with other individual MMPs.