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The U.S. Department of Agriculture (USDA) conducted a survey of domestic animal products including beef fat, pork fat, chicken fat, turkey fat, dairy cow fat, and siluriform fish muscle (i.e. catfish) to determine the current residue levels of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and non-ortho-polychlorinated biphenyls (no-PCBs). The following types of fat samples were analysed: beef from heifers and steers (referred to as “beef”), pork, chicken, turkey, dairy cow (referred to as “dairy cow”), and siluriform fish muscle, which includes catfish. The survey is conducted every 5 years. Fat samples for each slaughter class and siluriform fillets were collected from U.S. federally inspected slaughter facilities, processed, and analysed for 17 PCDD/Fs and 3 no-PCBs. Toxic equivalency concentrations were calculated using 2005 toxic equivalency factors and summed (sum-TEQ) for all slaughter classes. The sum-TEQs ranged from non-detect (nd) to 16.5 pg TEQ/g lipid. The median sum-TEQs, when nd = ½ LOD, for beef, pork, chicken, turkey, dairy cow, and siluriform fish were 0.48, 0.08, 0.07, 0.21, 0.38, and 0.62 pg TEQ/g lipid (wet weight for siluriform fish), respectively. A comparison of the current and previous surveys typically depicts a significant declining trend depending on the slaughter class (such as chicken and turkey), however, beef and pork fat median values slightly increased and decreased, respectively, but neither were significant compared to the previous survey. The congener patterns for some high concentration beef and pork fat samples suggested that pentachlorophenol treated wood continues to be a likely on-farm dioxin exposure source.

Semicarbazide (SEM) is the U.S. Food and Drug Administration's official marker for nitrofurazone use in food animals. The U.S. Department of Agriculture Food Safety and Inspection Service conducted a study to evaluate the source of SEM that was identified by a U.S. trading partner in a subset of chicken samples presented for inspection, even though nitrofurazone has been banned from use in U.S. food-producing animals since 2002. The study design included analyses to detect and quantify total and bound SEM in chicken collected from the eight U.S. establishments that were associated with the reported detection of SEM. Samples were collected immediately following evisceration, chilling, and cutting carcass into parts (cut-up). Although antimicrobial interventions (processes to reduce pathogen concentrations) are typically used at all three of these processing steps, the product contact time during chilling is significantly longer (hours versus seconds) than during evisceration and cut-up. In addition, parts were analyzed after 0, 10, 20, and 30 days of frozen storage. No postevisceration samples tested positive for SEM; however, most samples collected postchilling and after cut-up tested positive. The absence of SEM in postevisceration samples and detection in the subsequent postchilling samples and after the cut-up samples suggest that the detection of SEM in the sampled products is not indicative of preharvest nitrofurazone use and may be a result of postharvest processing in these establishments. HIGHLIGHTS

A versatile synthesis of 3-cyano-2-pyridones via a one-pot, four-component condensation of ethyl cyanoacetate, ketones, aldehydes, and ammonium acetate under very mild conditions has been developed. This method provides rapid access to this type of valuable heterocyclic compounds from readily available materials in a single operation.

Polyisoprenylation is a set of secondary modifications involving proteins whose aberrant activities are implicated in cancers and degenerative disorders. The last step of the pathway involves an ester-forming polyisoprenylated protein methyl transferase- and hydrolytic polyisoprenylated methylated protein methyl esterase (PMPMEase)-catalyzed reactions. Omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) have been linked with antitumorigeneis and tumorigenesis, respectively. PUFAs are structurally similar to the polyisoprenyl groups and may interfere with polyisoprenylated protein metabolism. It was hypothesized that PUFAs may be more potent inhibitors of PMPMEase than their more polar oxidative metabolites, the prostaglandins. As such, the relative effects of PUFAs and prostaglandins on PMPMEase could explain the association between cyclooxygenase-2 (COX-2) expression in tumors, the chemopreventive effects of the non-steroidal anti-inflammatory (NSAIDs) COX-2 inhibitors and PUFAs. PUFAs such as arachidonic (AA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids inhibited PMPMEase activity with Ki values of 0.12 to 3.7 μM. The most potent prostaglandin was 63-fold less potent than AA. The PUFAs were also more effective at inducing neuroblastoma cell death at physiologically equivalent concentrations. The lost PMPMEase activity in AA-treated degenerating cells was restored by incubating the lysates with COX-1 or COX-2. PUFAs may thus be physiological regulators of cell growth and could owe these effects to PMPMEase inhibition.

The polyisoprenylation pathway enzymes have been the focus of numerous studies to better understand the roles of polyisoprenylated proteins in eukaryotic cells and to identify novel targets against diseases such as cancer. The final step of the pathway is a reversible reaction catalyzed by isoprenyl carboxylmethyl transferase (icmt) whose products are then hydrolyzed by polyisoprenylated methylated protein methyl esterase (PMPMEase). Unlike the other pathway enzymes, the esterase has received little attention. We recently purified PMPMEase from porcine liver using an S-polyisoprenylated cysteine methyl ester substrate-dependent screening assay. However, no data is available showing its relative interaction with structurally diverse substrates. As such, its role as the putative endogenous PMPMEase has not been demonstrated. A series of substrates with S-alkyl substituents ranging from 2 to 20 carbons, including the two moieties found in polyisoprenylated proteins, were synthesized. Enzyme kinetics analysis revealed a 33-fold increase in affinity (K(M) values) from ethyl- (C-2, 505+/-63 microM), prenyl- (C-5, 294+/-25 microM), trans-geranyl- (C-10, 87+/-12 microM), trans, trans-farnesyl- (C-15, 29+/-2.2 microM) to all trans-geranylgeranyl- (C-20-, 15+/-2.7 microM) based analogs. Comparative molecular field analysis of the data yielded a cross-validated q(2) of 0.863+/-0.365 and a final R(2) of 0.995. Since the substrates with the S-trans, trans-farnesyl and S-all trans-geranylgeranyl moieties that occur in proteins show the highest affinity towards PMPMEase and are not hydrolyzed by the cholinesterases, the results suggest that polyisoprenylated proteins are the endogenous substrates of this esterase. The results suggest design strategies for high affinity and selective inhibitors of PMPMEase.

A versatile synthesis of 3-cyano-2-pyridones via a one-pot, four-component condensation of ethyl cyanoacetate, ketones, aldehydes, and ammonium acetate under very mild conditions has been developed. This method provides rapid access to this type of valuable heterocyclic compounds from readily available materials in a single operation.

Pancreatic cancer is the most deadly neoplasm with a 5-year survival rate of less than 6%. Over 90% of cases harbor K-Ras mutations, which are the most challenging to treat due to lack of effective therapies. Here, we reveal that polyisoprenylated methylated protein methyl esterase (PMPMEase) is overexpressed in 93% of pancreatic ductal adenocarcinoma. We further present polyisoprenylated cysteinyl amide inhibitors (PCAIs) as novel compounds designed with structural elements for optimal in vivo activities and selective disruption of polyisoprenylation-mediated protein functions. The PCAIs inhibited PMPMEase with Ki values ranging from 3.7 to 20 μM. The 48 h EC50 values for pancreatic cancer Mia PaCa-2 and BxPC-3 cell lines were as low as 1.9 μM while salirasib and farnesylthiosalicylamide were ineffective at 20 μM. The PCAIs thus have the potential to serve as effective therapies for pancreatic and other cancers with hyperactive growth signaling pathways mediated by Ras and related G-proteins.

Prostate cancer (CaP) is the most frequently diagnosed cancer in US men, with an estimated 236,590 new cases and 29,720 deaths in 2013. There exists the need to identify biomarkers/therapeutic targets for the early/companion diagnosis and development of novel therapies against the recalcitrant disease. Mutation and overexpression-induced abnormal activities of polyisoprenylated proteins have been implicated in CaP. Polyisoprenylated methylated protein methyl esterase (PMPMEase) catalyses the only reversible and terminal reaction of the polyisoprenylation pathway and may promote the effects of G proteins on cell viability. In this review, the potential role of PMPMEase to serve as a new drug target for androgen-insensitive CaP was determined. Specific PMPMEase activities were found to be 3.5- and 4.5-fold higher in androgen-sensitive 22Rv1 and androgen-dependent LNCaP and 1.5- and 9.8-fold higher in castration-resistant DU 145 and PC-3 CaP cells compared to normal WPE1-NA22 prostate cells. The PMPMEase inhibitor, L-28, induced apoptosis with EC50 values ranging from 1.8 to 4.6 μM. The PMPMEase activity in the cells following treatment with L-28 followed a similar profile, with IC50 ranging from 2.3 to 130 μM. L-28 disrupted F-actin filament organisation at 5 μM and inhibited cell migration 4-fold at 2 μM. Analysis of a CaP tissue microarray for PMPMEase expression revealed intermediate, strong, and very strong staining in 94.5% of the 92 adenocarcinoma cases compared to trace and weak staining in the normal and normal-adjacent tissue controls. The data are an indication that effective targeting of PMPMEase through the development of more potent agents may lead to the successful treatment of metastatic CaP.

The involvement of hyperactive polyisoprenylated proteins in cancers has stimulated the search for drugs to target and suppress their excessive activities. Polyisoprenylated methylated protein methyl esterase (PMPMEase) inhibition has been shown to modulate polyisoprenylated protein function. For PMPMEase inhibition to be effective against cancers, polyisoprenylated proteins, the signaling pathways they mediate and/or PMPMEase must be overexpressed, hyperactive and be involved in at least some cases of cancer. PMPMEase activity in lung cancer cells and its expression in lung cancer cells and cancer tissues were investigated. PMPMEase was found to be overexpressed and significantly more active in lung cancer A549 and H460 cells than in normal lung fibroblasts. In a tissue microarray study, PMPMEase immunoreactivity was found to be significantly higher in lung cancer tissues compared to the normal controls (p < 0.0001). The mean scores ± SEM were 118.8 ± 7.7 (normal), 232.1 ± 25.1 (small-cell lung carcinomas), 352.1 ± 9.4 (squamous cell carcinomas), 311.7 ± 9.8 (adenocarcinomas), 350.0 ± 24.2 (papillary adenocarcinomas), 334.7 ± 30.1 (adenosquamous carcinomas), 321.9 ± 39.7 (bronchioloalveolar carcinomas), and 331.3 ± 85.0 (large-cell carcinomas). Treatment of lung cancer cells with L-28, a specific PMPMEase inhibitor, resulted in concentration-dependent cell death (EC50 of 8.5 μM for A549 and 2.8 μM for H460 cells). PMPMEase inhibition disrupted actin filament assembly, significantly inhibited cell migration and altered the transcription of cancer-related genes. These results indicate that elevated PMPMEase activity spur cell growth and migration, implying the possible use of PMPMEase as a protein biomarker and drug target for lung cancer.

Inhibition of PMPMEase, a key enzyme in the polyisoprenylation pathway, induces cancer cell death. In this study, purified PMPMEase was inhibited by the chemopreventive agent, curcumin, with aKiof 0.3 μM (IC50= 12.4 μM). Preincubation of PMPMEase with 1 mM curcumin followed by gel-filtration chromatography resulted in recovery of the enzyme activity, indicative of reversible inhibition. Kinetics analysis with N-para-nitrobenzoyl-S-trans,trans-farnesylcysteine methyl ester substrate yieldedKMvalues of 23.6 ± 2.7 and 85.3 ± 15.3 μM in the absence or presence of 20 μM curcumin, respectively. Treatment of colorectal cancer (Caco2) cells with curcumin resulted in concentration-dependent cell death with an EC50of 22.0 μg/mL. PMPMEase activity in the curcumin-treated cell lysate followed a similar concentration-dependent profile with IC50of 22.6 μg/mL. In colorectal cancer tissue microarray studies, PMPMEase immunoreactivity was significantly higher in 88.6% of cases compared to normal colon tissues(P<0.0001). The mean scores ± SEM were 91.7 ± 11.4 (normal), 75.0 ± 14.4 (normal adjacent), 294.8 ± 7.8 (adenocarcinoma), and 310.0 ± 22.6 (mucinous adenocarcinoma), respectively. PMPMEase overexpression in colorectal cancer and cancer cell death stemming from its inhibition is an indication of its possible role in cancer progression and a target for chemopreventive agents.

The polyisoprenylation pathway incorporates a reversible step that metabolizes polyisoprenylated methylated proteins from the ester to the carboxylate form. Polyisoprenylated protein methyl transferase (PPMTase) catalyses the esterification whereas polyisoprenylated methylated protein methyl esterase (PMPMEase) hydrolyzes them. Significant changes in the balance between the two enzymes may alter polyisoprenylated protein function possibly resulting in disease. Previous studies show that PMPMEase is the serine hydrolase, Sus scrofa carboxylesterase. Its susceptibility to the nonspecific serine hydrolase inhibitor, phenylmethylsulfonyl fluoride (PMSF) paved the way for its use as a prototypical compound to design and synthesize a series of putative high affinity specific inhibitors of PMPMEase. Pseudo first-order kinetics revealed an over 680-fold increase in k(obs)/[I] values from PMSF (6 M(-1)-1s(-1)), S-phenyl (L-50, 180 M(-1)s(-1)), S-benzyl (L-51, 350 M(-1)s(-1)), S-trans, trans-farnesyl (L-28, 2000 M(-1)s(-1)), to S-trans-geranylated (L-23, 4100 M(-1)s(-1)) 2-thioethanesulfonyl fluorides. C10 S-alkyl substitution revealed a k(obs)/[I] value (1800 M(-1)s(-1)) that was 298 times greater than that for PMSF. The compounds induced the degeneration of human neuroblastoma SH-SY5Y cells with EC(50) values of 49, 130 and1000 µM for L-28, L-23 and PMSF, respectively. The increased affinity with the polyisoprenyl derivatization is consistent with the observed substrate specificity and the reported hydrophobic nature of the active site. These results suggest that (1) PMPMEase is a key enzyme for polyisoprenylated protein metabolism, (2) regulation of its activity is essential for maintaining normal cell viability, (3) abnormal activities may be involved in degenerative diseases and cancers and (4) its specific inhibitors may be useful in combating cancers.

Polyisoprenylated proteins (PPs) methylation by polyisoprenylated protein methyl transferase (PPMTase) is counteracted by polyisoprenylated methylated protein methyl esterase (PMPMEase). This is the only reversible step of the polyisoprenylation pathway as the relative amounts of the acid and ester forms are determined by the two competing reactions. Since PMPMEase and PPMTase may influence both the structural/functional conformations of PPs, a thorough study of these enzymes is essential to our understanding of the structural/functional features of PPs. PMPMEase has been reported under such pseudonyms as human carboxylesterase 1 (hCE1) because of its apparent broad substrate spectrum. The current study aimed to show the complementarity between its active site and the polyisoprenylated substrates that it metabolizes. Kinetics analysis was conducted with N-, S- and O-substituted substrates using porcine liver PMPMEase and docking analysis using Arguslab. Consistent with the biochemical analysis, the S-ethyl analog yielded an AScore binding energy of -11.32 compared to -13.48, -14.88, -16.15, and -16.81 kcal/mol for S-prenyl (C-5), S-trans-geranyl (C-10), S-trans,trans-farnesyl (C-15) and S-all trans-geranylgeranyl (C-20), respectively. The all trans-geranylgeranyl moiety provides the optimal size for active site interactions. The data reveal that the trans,trans-farnesyl and all trans-geranylgeranyl groups, which are reminiscent of endogenous PPs modifications, have the highest affinity for PMPMEase. Since PPs such as monomeric G-proteins are oncogenic, PMPMEase may be viewed as a viable target for anticancer drug development. The analyses reveal the important structural elements for the design of specific PMPMEase inhibitors to serve in the modulation of oncogenic PPs activities. The results also show that hCE1's repertoire of substrates extends beyond xenobiotics to include PPs as its endogenous substrates.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most deadly neoplasms due to lack of effective targeted therapies. This vacuum stems from the fact that over 90% of pancreatic cancer cases harbor the K-Ras mutation which has been very challenging to drug. The numerous efforts to drug oncogenic Ras have involved inhibiting the post-translational modifications that are essential to its proliferative activity. These drug discovery efforts have largely ignored a critical step in the metabolism of Ras and related proteins involving polyisoprenylated methylated protein methyl esterase (PMPMEase). The objectives of these studies were to test the hypothesis that PMPMEase hyperactivity promotes pancreatic cancer progression. The potential for polyisoprenylated cysteinyl amide inhibitors (PCAIs) of PMPMEase as potential anticancer agents was evaluated against PDAC cell lines MIAPaCa-2 with K-Ras mutation and BxPC-3 with wild type K-Ras for their effects on cell viability, migration, and cytoskeletal organization. The PCAIs inhibited the viability of MIAPaCa-2 and BxPC-3 cells with 48 h EC50 values as low as 1.9 and 3.2 µM, respectively. Erlotinib and Salirasib (FTS) were ineffective at concentrations in excess of 20 µM. The PCAIs also inhibited MIAPaCa-2 cell migration by up to 50% at 0.5 µM and disrupted F-actin organization at 5 µM. Further investigations showed that at the EC50 concentrations the PCAIs caused the pancreatic cancer cells to die by apoptosis. The cell cycle analysis for MIAPaCa-2 cells showed that the PCAIs block the cells growth at G0/G1 phase. The results showed that the PCAIs are over 10-fold more effective than Salirasib and Erlotinib against PDAC cell viability. Furthermore, their effects against cell migration and actin filament organization suggest their potential for treating tumor growth and metastasis in not only PDAC but other cancers with oncogenic Ras. Citation Format: Augustine T. Nkembo, Byron J. aguilar, Randolph Duverna, Felix Amissah, Nazarius S. Lamango. Polyisoprenylated cysteinyl amide inhibitors of PMPMEase inhibit pancreatic cancer cell viability and migration: Implications for pancreatic cancer therapy. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B89.

Pancreatic cancer harbors the highest reported rates of K-Ras mutations, lacks effective therapies and is the most challenging to treat. It is thus the most deadly neoplasm with a 5-year survival rate of only 6%. We reveal here that polyisoprenylated methylated protein methyl esterase (PMPMEase) is overexpressed in 93% of pancreatic duct adenocarcinoma. We further present novel compounds with the potential to serve as effective targeted therapies for pancreatic and other cancers with hyperactive growth signaling pathways mediated by Ras and related G-proteins. The design and synthesis of the polyisoprenylated cysteinyl amide inhibitors (PCAIs) of PMPMEase incorporate three key elements to obtain compounds to selectively disrupt polyisoprenylation-mediated protein-protein interactions; the farnesyl group for high affinity interactions, a substituted amide bioisostere of the scissile ester bond of the endogenous substrates and an ionizable appendage group designed to mitigate the excessive hydrophobicity of the farnesyl cysteinyl amide that constitutes the pharmacophore. The PCAIs inhibited PMPMEase with Ki values ranging from 3.7 to 20 μM. The 48 h EC50 values for pancreatic cancer Mia PaCa-2 and BxPC-3 cell lines were as low as 1.9 μM while Salirasib (farnesylthiosalicylic acid, FTS) and farnesylthiosalicylamide were not effective even at 20 μM. The PCAIs thus have the potential to serve as a novel class of targeted therapies for cancers with hyperactive G-proteins and the growth factor receptors whose signals they transmit. Citation Format: Nazarius S. Lamango, Byron J. Aguilar, Augustine T. Nkembo, Randolph Duverna, Felix Amissah, Rosemary A. Poku, Seth Y. Ablordeppey. Polyisoprenylated cysteinyl amides as targeted small molecule anti-Ras therapies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-14. doi:10.1158/1538-7445.AM2014-LB-14

PURPOSE: Excessive epidermal growth factor receptor (EGFR) signaling and/or hyperactivities of polyisoprenylated monomeric G-proteins of the Ras superfamily are frequently associated with various human cancers including lung cancer. While mutations or overexpression of these proteins are responsible for their tumorigenic effects, secondary modifications of the monomeric G-proteins involving polyisoprenylation and eventual methylation are required for their normal as well as the pathological activities. Despite the use of anti-EGFR targeted therapies, prognosis is still poor for patients whose tumors harbor the defective receptor. The need to find novel biomarkers for early/companion diagnosis and targets for the development of novel therapies remains strong. Since polyisoprenylated methylated protein methyl esterase (PMPMEase) is a critical enzyme of the polyisoprenylation pathway whose inhibition causes cancer cell death, the objective was to determine whether it is overexpressed and/or hyperactive in lung cancers in which case, it might serve as a possible diagnostic biomarker or drug target. METHODS: Tissue microarrays (TMAs) consisting of 416 cores from 416 separate normal lung and lung cancer cases were analyzed for PMPMEase expression. Also, PMPMEase activities in fresh frozen lung cancer tissues were determined. PMPMEase expression and activities in normal lung fibroblasts (WI-38) as well as lung cancer A549 and H460 cells were also determined. The effect of specific PMPMEase inhibition with L-28 on cell viability was also tested. RESULTS: Significantly more intense PMPMEase immunoreactivity was observed in 88.3% of the 416 cases in the lung cancer compared to the normal controls (p < 0.0001). The mean scores ± SEM were 118.8±7.7 (normal), 232.1±25.1 (small-cell lung carcinomas), 352.1±9.4 (squamous cell carcinomas), 311.7±9.8 (adenocarcinomas), 350.0±24.2 (papillary adenocarcinomas), 334.7±30.1 (adenosquamous carcinomas), 321.9±39.7 (bronchioloalveolar carcinomas), and 331.3±85.0 (large-cell carcinomas). PMPMEase was significantly more active in five of the ten lung cancer cases compared to normal adjacent tissues (p < 0.001). The specific PMPMEase activity in A549 (5.2 ± 0.10 mmol/h/mg of protein) and H460 (4.8 ± 0.05 mmol/h/mg of protein) cells was 1.6- and 1.5-fold higher than in WI-38 cells (3.2 ± 0.04 mmol/h/mg of protein), respectively. Exposure of the cells to L-28, led to lung cancer cell death, with EC50 of 8.5 x 10-6 M for A549 and 2.8 x 10-5 M for H460 cells. CONCLUSION: These results implicate PMPMEase overexpression and hyperactivity in lung cancer progression, implying that PMPMEase could serve as a marker for diagnosis and target for lung cancer therapy. Citation Format: Felix Amissah, Randolph Duverna, Byron J. Aguilar, Rosemary A. Poku, Lamango S. Lamango. Polyisoprenylated methylated protein methyl esterase as a potential therapeutic target and biomarker in lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4357. doi:10.1158/1538-7445.AM2013-4357

Lack of targeted, more effective therapies for triple negative breast cancer (TNBC) has necessitated the continuous search for protein biomarkers and targets for novel therapeutic development. Polyisoprenylated monomeric G-proteins such as Ras and Rab are hyperactive in about 30% of cancers and have profound effects on cell growth. Inhibition of polyisoprenylated methylated protein methyl esterase (PMPMEase) causes the death of cancer cells thus suggesting that hyperactivity of PMPMEase may result in cancers. The current studies were to address the hypothesis that PMPMEase activity is elevated in breast cancer. PMPMEase activity was determined in breast tumors and normal adjacent tissues (NAT) in snap-frozen tissues from 10 breast cancer cases. Five- to over 20-fold increases in PMPMEase activity compared to the respective NATs were detected in 8 of the 10 cases. Specific activities ranging from 0.25 ± 0.02 to 1.58 ± 0.07 nmol/h/mg were detected in the tumors compared to 0.09 nmol/h/mg or less in the NATs. Breast cancer tissue microarrays (TMAs) consisting of 125 independent breast cancer cases and NATs were analyzed for PMPMEase expression by immunohistochemistry and scored for their immunoreactive staining intensities. Intermediate to very strong immunoreactivities were detected in the breast cancer TMA core slices compared to only trace staining in those of the NATs. The mean scores ± SEM ranged from 310 ± 21, 290 ± 7, 350 ± 15, 360 ± 18, 390 ± 14, 360 ± 29, 390 ± 13, 275 ± 10 to 65 ± 10 for TNBC, ER(+), ER(+)PR(+), ER(+)HER2(+), ER(+)PR(+)HER2(+), PR(+)HER2(+), HER2(+), PR(+) and NATs, respectively. These results show that PMPMEase overexpression in breast cancer may explain the increased enzymatic activity in the tumors. PMPMEase enzymatic activity and/or immunoreactivity may serve as diagnostic procedures for breast cancer. PMPMEase may also serve as valid target for anticancer drug development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1843. doi:1538-7445.AM2012-1843

Polyisoprenylated methylated protein methylesterase (PMPMEase) is a little explored enzyme of the polyisoprenylation (isoprenylation) pathway (PP). Polyisoprenylation is a post-translational modification that is important for the function of such proteins as the heterotrimeric G proteins, Ras and Rab families of proteins. PMPMEase hydrolyzes a methyl ester formed by the S-adenosyl-L-methionine (SAM)-dependent enzyme, polyisoprenylated protein methyltransferase (PPMTase). PMPMEase's regulatory role is suggested by the fact it hydrolyzes the ester products of PPMTase. PMPMEase is a serine hydrolase, a class of hydrolytic enzymes that attack electron-deficient carbons of amide and ester bonds with the alkoxide ion of their catalytic serines. Because alkylating agents function through an aziridinium ion with an electron-deficient carbon atom, it is hypothesized that they may inhibit PMPMEase through active site adduct formation. Alkylating agents have been suspected of having other mechanisms of action against cells besides impeding replication via the cross-linking of DNA strands. The crystal structure of the human form of PMPMEase, human carboxylesterase 1, with tamoxifen bound to its active site has been solved, also suggesting its possible inhibition. Polyisoprenylated proteins are involved in processes that regulate cell viability. Mutations and overexpression of such proteins are implicated in about 30% of cancers. This study considers the possibility that the agents also reduce malignant cell growth by disrupting growth signaling pathways. Cyclophosphamide is an organophosphorous alkylating agent. Organophosphorous compounds irreversibly inhibit PMPMEase and other serine esterases. Busulfan, cyclophosphamide, mechlorethamine and tamoxifen were tested for porcine liver PMPMEase inhibition. At 1 mM concentrations, cyclophosphamide, mechlorethamine and tamoxifen inhibited 62, 35 and 55% of the PMPMEase activity. The results show that the anticancer drugs may owe part of their pharmacological activity to their modulation of oncogenic polyisoprenylated protein function through their inhibition of PMPMEase. Citation Information: Cancer Res 2009;69(23 Suppl):C71.