Molecularly targeted therapies in cancer: a guide for the nuclear medicine physician.

Molecular imaging continues to influence every aspect of cancer care including detection, diagnosis, staging and therapy response assessment. Recent advances in the understanding of cancer biology have prompted the introduction of new targeted therapy approaches. Precision medicine in oncology has led to rapid advances and novel approaches optimizing the use of imaging modalities in cancer care, research and development. This article focuses on the concept of targeted therapy in cancer and the challenges that exist for molecular imaging in cancer care.

Epitope complementarity and idiotypic interactions: a study of idiotypic-like interactions between anti-cytidine and anti-guanosine A/J mouse monoclonal antibodies--I. Characterization of these interactions.

Idiotypic-like interactions between mAbs directed against cytidine (Cyd) or guanosine (Guo) nucleosides were characterized. These mAbs, Cyd-1 (IgG2b, kappa), Guo-1 (IgG1, kappa) and Guo-2 (IgG1, kappa) were derived from splenocytes of A/J mice immunized with Cyd-KLH or Guo-KLH and recognized the nucleoside base moieties involved in hydrogen bonding. The interactions between Guo-1 or Guo-2 and Cyd-1 involved cross-reactive or distinct-but-neighboring paratope-associated idiotopes. These interactions were characterized by KD values of 4.6 x 10(-6) and 1.8 x 10(-6)M, respectively. The three anti-nucleoside mAbs exhibited Ab2 beta properties and manifested epibody (Ab2 epsilon) activity towards ssDNA. We compared these idiotypic-like reactivities with the anti-idiotypic activity of an intentionally induced IgG1, kappa anti-idiotype mAb prepared with splenocytes from A/J mice immunized with Cyd-1. This Ab2 antibody which bound to Cyd-1 with a KD of 1.1 x 10(-9) M, manifested an Ab2 gamma activity, i.e. it recognized a paratope-associated idiotope on Cyd-1 without exhibiting Ab2 beta properties. In addition, the anti-(Cyd-1) completely inhibited (Cyd-1)-(Guo-1) and (Cyd-1)-(Guo-2) interactions.

Intramuscular variation in beef tenderness.

Intramuscular variation of beef tenderness was investigated in three muscles: Semitendinosus (ST), Triceps brachii (TB) and Rectus femoris (RF). The aim of the study was to evaluate a French process (called "affranchi") used to improve meat tenderness. Two groups of 72 muscles (24 ST, 24 TB and 24 RF) aged 10 and 14 days respectively, were assessed for tenderness by sensory analysis and compression measurements. For all three muscles, sensory evaluation showed a significant intramuscular variation in tenderness with a longitudinal gradient. Compression measurements on the raw meat showed similar results but of reduced accuracy. It appears that the process "affranchi" which consists in removing a part of the muscle, is very useful to improve meat tenderness and could be used by meat processors. The low relationship between compression measurements and sensory evaluation suggest compression measurements on raw meat are not suitable to predict meat tenderness as perceived by consumers.

Objective measurement of veal color for classification purposes.

2300 veal calves were used to compare the ability of chromameters to measure the veal meat color on-line and to develop a relationship between instrumental and visual assessments to predict the color score according to the EC-system of classification. The meat color was assessed subjectively by 3 trained judges and objectively by 2 chromameters CR300 and CR310, 45 min post mortem on the Rectus abdominis, on the external side, skin having been removed. R(2) values reached 0.70 for the CR300 and 0.75 for the CR310. The equations of prediction classified correctly up to 87% of carcasses. These data indicate that chromameters (Minolta CR310 and CR300) can be used on-line to measure objectively veal meat color at the end of the slaughterline.

Assessment of meat fat content using dual energy X-ray absorption.

Fat content is an essential component of meat quality. Fat content and fatty tissue content were determined by dual energy X-ray absorption on 3 types of meat: pork meat (mixture of longissimus dorsi and fat) and beef meat (longissimus dorsi and pectoralis profondus). The measurements were carried out with a medical densitometer, the SOPHOS L-XRA usually used for osteodensitometry. The results from the dual energy X-ray absorption and the chemical analysis were compared and the correlations were good to very good (R(2) values from 0.7 to 0.97). The residual standard deviations were in the range 2.75-4.89%. The routine use of dual energy X-ray absorption would though suppose a previous calibration.

Fine mapping of quantitative trait loci underlying sensory meat quality traits in three French beef cattle breeds.

Improving the traits that underlie meat quality is a major challenge in the beef industry. The objective of this paper was to detect QTL linked to sensory meat quality traits in 3 French beef cattle breeds. We genotyped 1,059, 1,219, and 947 young bulls and their sires belonging to the Charolais, Limousin, and Blonde d'Aquitaine breeds, respectively, using the Illumina BovineSNP50 BeadChip (Illumina Inc., San Diego, CA). After estimating relevant genetic parameters using VCE software, we performed a linkage disequilibrium and linkage analysis on 4 meat traits: intramuscular fat content, muscle lightness, shear force, and tenderness score. Heritability coefficients largely ranged between 0.10 and 0.24; however, they reached a maximum of 0.44 and 0.50 for intramuscular fat content and tenderness score, respectively, in the Charolais breed. The 2 meat texture traits, shear force and tenderness score, were strongly genetically correlated (-0.91 in the Charolais and Limousin breed and -0.86 in the Blonde d'Aquitaine breed), indicating that they are 2 different measures of approximately the same trait. The genetic correlation between tenderness and intramuscular fat content differed across breeds. Using a significance threshold of 5 × 10(-4) for QTL detection, we found more than 200 significant positions across the 29 autosomal chromosomes for the 4 traits in the Charolais and Blonde d'Aquitaine breeds; in contrast, there were only 78 significant positions in the Limousin breed. Few QTL were common across breeds. We detected QTL for intramuscular fat content located near the myostatin gene in the Charolais and Blonde d'Aquitaine breeds. No mutation in this gene has been reported for the Blonde d'Aquitaine breed; therefore, it suggests that an unknown mutation could be segregating in this breed. We confirmed that, in certain breeds, markers in the calpastatin and calpain 1 gene regions affect tenderness. We also found new QTL as several QTL on chromosome 3 that are significantly associated with meat tenderness in the Blonde d'Aquitaine breed. Overall, these results greatly contribute to the goal of building a panel of markers that can be used to select animals of high meat quality.

miR-491-5p-induced apoptosis in ovarian carcinoma depends on the direct inhibition of both BCL-XL and EGFR leading to BIM activation.

We sought to identify miRNAs that can efficiently induce apoptosis in ovarian cancer cells by overcoming BCL-X(L) and MCL1 anti-apoptotic activity, using combined computational and experimental approaches. We found that miR-491-5p efficiently induces apoptosis in IGROV1-R10 cells by directly inhibiting BCL-X(L) expression and by inducing BIM accumulation in its dephosphorylated form. This latter effect is due to direct targeting of epidermal growth factor receptor (EGFR) by miR-491-5p and consequent inhibition of downstream AKT and MAPK signalling pathways. Induction of apoptosis by miR-491-5p in this cell line is mimicked by a combination of EGFR inhibition together with a BH3-mimetic molecule. In contrast, SKOV3 cells treated with miR-491-5p maintain AKT and MAPK activity, do not induce BIM and do not undergo cell death despite BCL-XL and EGFR downregulation. In this cell line, sensitivity to miR-491-5p is restored by inhibition of both AKT and MAPK signalling pathways. Altogether, this work highlights the potential of miRNA functional studies to decipher cell signalling pathways or major regulatory hubs involved in cell survival to finally propose the rationale design of new strategies on the basis of pharmacological combinations.

Effects of polymorphisms in the calpastatin and µ-calpain genes on meat tenderness in 3 French beef breeds.

The objectives of the study were to evaluate allelic frequencies and to test the association of polymorphisms in the calpastatin (CAST) and µ-calpain (CAPN1) genes with meat tenderness in 3 French beef breeds. A total of 1,114 Charolais, 1,254 Limousin, and 981 Blonde d'Aquitaine purebred young bulls were genotyped for 3 SNP in the CAST gene and 4 SNP in the CAPN1 gene. Two of these markers, 1 in each gene, can be found in Australian or American commercial genetic tests. Others have previously been reported in American studies or are newly evidenced SNP. The quantitative traits studied were Warner-Bratzler shear force and a tenderness score evaluated by trained sensory panels. All the SNP were informative in the 3 breeds. Associations of individual markers or haplotypes with traits were analyzed. The results differed in the 3 breeds. The G allele of a CAST marker (position 97574679 on Btau4.0) was found to exert a significant effect on the shear force (+0.18 phenotypic SD; RSD) and tenderness score (-0.22 RSD) in the Blonde d'Aquitaine breed. In the same breed, this marker was associated with another CAST SNP (position 97576054 on Btau4.0) such that the GA haplotype appeared to be associated with tougher meat. Two CAPN1 markers (positions 45221250 and 45241089 on Btau4.0) had a significant effect on both traits in the Charolais breed (from |0.11| to |0.25| RSD). In the same breed, these markers were associated with another CAPN1 SNP (position 45219395 on Btau4.0) such that the ACA and AGG haplotypes appeared to be associated with a tender meat and a tougher meat, respectively. Consequently, the present results indicate that the effects of the markers studied are breed-specific and cannot be extended to all Bos taurus breeds. Further studies are also required to identify other more appropriate markers for French beef breeds.

The two mutations, Q204X and nt821, of the myostatin gene affect carcass and meat quality in young heterozygous bulls of French beef breeds.

The availability of genetic tests to detect different mutations in the myostatin gene allows the identification of heterozygous animals and would warrant the superiority of these animals for slaughter performance if this superiority is confirmed. Thus, 2 mutations of this gene, Q204X and nt821, were studied in 3 French beef breeds in the program Qualvigène. This work was done with 1,114 Charolais, 1,254 Limousin, and 981 Blonde d'Aquitaine young bulls from, respectively, 48, 36, and 30 sires and slaughtered from 2004 to 2006. In addition to the usual carcass traits recorded at slaughter (e.g., carcass yield, muscle score), carcass composition was estimated by weighing internal fat and dissecting the 6th rib. The muscle characteristic traits analyzed were lipid and collagen contents, muscle fiber section area, and pH. Regarding meat quality, sensory qualities of meat samples were evaluated by a taste panel, and Warner-Bratzler shear force was measured. Deoxyribonucleic acid was extracted from the blood samples of all calves, the blood samples of 78% of the dams, and the blood or semen samples of all the sires. Genotypes were determined for 2 disruptive mutations, Q204X and nt821. Analyses were conducted by breed. The superiority of carcass traits of calves carrying one copy of the mutated allele (Q204X or nt821) over noncarrier animals was approximately +1 SD in the Charolais and Limousin breeds but was not significant in the Blonde d'Aquitaine. In the Charolais breed, for which the frequency was the greatest (7%), young bulls carrying the Q204X mutation presented a carcass with less fat, less intramuscular fat and collagen contents, and a clearer and more tender meat than those of homozygous-normal cattle. The meat of these animals also had slightly less flavor. Also in the Charolais breed, 13 of 48 sires were heterozygous. For each sire, the substitution effect of the wild allele by the mutant allele was approximately +1 SD for carcass conformation and yield, showing that the estimate of the substitution effect was independent of family structure, as it ought to be for a causal mutation. These results illustrate the challenge of using genetic tests to detect animals with the genetic potential for greater grades of carcasses and meat quality.

Anti-RhoA and anti-RhoC siRNAs inhibit the proliferation and invasiveness of MDA-MB-231 breast cancer cells in vitro and in vivo.

Overexpression of RhoA or RhoC in breast cancer indicates a poor prognosis, due to increased tumor cell proliferation and invasion and tumor-dependent angiogenesis. Until now, the strategy of blockage of the Rho-signaling pathway has used either GGTI or HMG-CoA reductase inhibitors, but they are not specific to RhoA or RhoC inhibition. In this study, a new approach with anti-RhoA and anti-RhoC siRNAs was used to inhibit specifically RhoA or RhoC synthesis. Two transfections of either RhoA or RhoC siRNA (8.5 nM) into MDA-MB-231 human breast cancer cells or HMEC-1 endothelial cells induced extensive degradation of the target mRNA and led to a dramatic decrease in synthesis of the corresponding protein. In vitro, these siRNAs inhibited cell proliferation and invasion more effectively than conventional blockers of Rho cell signaling. Finally, in a nude mouse model, intratumoral injections of anti-RhoA siRNA (100 microl at 85 nM) every 3 days for 20 days almost totally inhibited the growth and angiogenesis of xenografted MDA-MB-231 tumors. One may infer from these observations that specific inhibition of the Rho-signaling pathway with siRNAs represents a promising approach for the treatment of aggressive breast cancers.

Purification and partial characterization of an amino acid alpha,beta- dehydrogenase, L-tryptophan 2',3'-oxidase from Chromobacterium violaceum.

L-Tryptophan 2',3'-oxidase is a novel enzyme that specifically catalyzes the alpha,beta-dehydrogenation of L-tryptophan derivatives. It was extracted from Chromobacterium violaceum and purified 108-fold to apparent homogeneity with a 34% overall recovery. The molecular weight of the native enzyme is approximately 680,000, and its isoelectric point is nearly equal to 4. SDS-polyacrylamide gel electrophoresis showed that the enzyme is composed of two components with molecular weight of approximately 74,000 and 14,000. It also contains a noncovalently bound heme prosthetic group, as judged from a typical spectrum showing maxima at 427, 530, and 560 nm. The catalyzed reaction is completed without side-product formation over a broad pH range comprised between 3 and 8. The enzyme is reoxidized at the expense of molecular oxygen by producing one molecule of H2O2. Kinetic parameters for modification of N-acetyl-L-tryptophanamide were determined (Km = 19.5 microM; kcat = 45.2 s-1). A Hill coefficient of about 1 suggests the absence of any cooperative effect. As inferred from both its kinetic and stability optimal conditions, L-tryptophan 2',3'-oxidase constitutes a promising tool for chemical modification of tryptophanyl side chain in peptides and proteins.

Role of CD4+ and CD8+ cell subsets during amplification of natural T cell activity against IgG2ab in Igha mice and during induction of IgG2ab allotype suppression in Igha/b mice.

Transfer into F1 Igha/b mice of splenocytes from Igha mice sensitized once against B cells from an Ighb congenic strain induces total, chronic, and IgG2ab (IgG2a of the Ighb haplotype)-specific allotype suppression in these recipients. We previously demonstrated that both the CD4+ and CD8+ T subsets were necessary for inducing suppression, but that CD8+, cells by themselves were sufficient for maintaining suppression. We have studied the suppression induction capacity of different mixtures of CD4+ and CD8+ subsets obtained by in vitro cytotoxic treatment of T splenocytes from normal or sensitized Igha mice, and we have established that suppression induction requires the cooperation between CD4+ and CD8+ populations, both of which have to be IgG2ab specific. In addition, Igha mice were sensitized in the absence of CD4+ or CD8+ cells by in vivo cytotoxic treatment performed before and after the sensitization in order to obtain an IgG2ab-specific CD4+ population that has arisen in the absence of CD8+ cells, and vice versa. We found that only IgG2ab-specific CD4+ cells from anti-CD8-treated mice (T'sens CD4+) had the ability to induce suppression in F1 Igha/b hosts. Nevertheless, the real effector cells in this suppression model display the CD8+ phenotype, as in vivo cytotoxic anti-CD8 treatment of Igha/b recipients of T'sens CD4+ abrogates the suppression induction capacity. Taken together, these results show that T'sens CD4+ have an important capacity to recruit CD8+ anti-IgG2ab effector cells from precursors that have been transferred with them into Igha/b hosts. These precursors are actually derived from the T'sens CD4+ cell preparation, because we have recently demonstrated that suppression is maintained by donor T cells throughout the recipient's life. CD4+ cells can have their anti-IgG2ab activity amplified only by means of target cells (i.e., B cells from Ighb congenic mice), whereas, in the absence of CD4+ cells, and despite the presence of target cells, CD8+ cells seem unable to acquire this amplified activity.

Inhibition of IgG2ab production by Ig allotype-specific T cells can Be mediated without T-B cell contact.

The growth of IgG2ab-producing CB101 myeloma cells, subcutaneously or intraperitoneally inoculated into histocompatible BALB/c Igha mice sensitized against this Ig allotype, was delayed by 2-4 weeks compared to normal mice. While IgG2ab production was detected in the sera of 75-100% of normal mice, it was irreversibly inhibited in 100% of sensitized mice. IgG2ab suppression (IgG2ab sup) was also systematically obtained in sensitized but not normal recipients, implanted ip with a 0.1-micrometer-pore diffusion chamber (DC) containing CB101 cells. This time, the specific IgG2ab sup was reversible in vitro in the absence of anti-IgG2ab T cells. Adoptive transfer, of unfractionated or T but not B splenocytes from their sensitized counterparts into normal mice, 1 day before DC implantation, induced IgG2ab sup as well. These results indicate that, in these experimental circumstances, IgG2ab sup can also be mediated by diffusible suppressive factors produced by the effector T cells, without direct T-B-cell contact.

T cell-induced Ig allotypic suppression in mice. Basis for emergence or tolerization, during the perinatal period, of natural T cells specific to the IgG2ab allotype.

We have previously described an anti-IgG2ab T cell activity in normal Igha/a mice. Their congenic partner at the Igh locus (Ighb/b) and Igha/b hybrids bred from them, do not display this T cell activity but express IgG2ab. As these mice are supposed to possess the same genetic elements related to this potential T cell repertoire, only somatic selection mechanisms could be responsible for their different behavior. In this study, we investigated the basis for the emergence (in Igha/a mice) or tolerization (in Ighb/b-congenic mice and in Igha/b hybrids) of these natural anti-IgG2ab T cells. Stringent perinatal B cell deprivation in Ighb/b and Igha/b mice resulted in the emergence of anti-IgG2ab T cells, as these individuals could be subjected to autoimmune, T cell-mediated IgG2ab suppression. Furthermore, the acquisition of anti-IgG2ab T cell activity was drastically reduced in Igha/a mice, perinatally exposed to IgG2ab; thus, the presence of this allotype leads to tolerization of these specific T cells.

New insights into the actions of bisphosphonate zoledronic acid in breast cancer cells by dual RhoA-dependent and -independent effects.

Zoledronic acid (ZOL) is a nitrogen-containing bisphosphonate and its use in reducing osteoporosis and cancer-induced osteolysis is increasing. Recent findings indicated that ZOL has a direct effect on cancer cells. In this study, the effect of ZOL was examined on the aggressive MDA-MB-231 breast cancer cell line. ZOL induces an important inhibition of cell invasion at low concentrations (1 microM). This is not explained by modifications of proteases involved in cell invasiveness (matrix metalloproteinases and urokinase-type plasminogen activator), but by a disorganisation of actin cytoskeleton due to RhoA inhibition related to its defective prenylation as it was reversed by geranylgeraniol (GGOH) and mimicked by the Rho selective inhibitor C3 exoenzyme. In addition, ZOL inhibits the chemotactic effect induced by stromal cell-derived factor 1(SDF-1), a chemokine greatly involved in cancer metastasis to bone. This effect is related to both reduction of cell motility induced by RhoA inhibition and to a decreased expression of CXCR-4, the SDF-1 receptor. Finally, ZOL reduces Cox-2 expression and, consequently, the secretion of prostaglandins E2 (PGE2) in a RhoA-independent manner. This inhibition could contribute to bone protection in breast cancers because PGE2 stimulates osteoclast-mediated bone resorption. In summary, new insights in the mechanism of ZOL action on aggressive breast cancer cells are demonstrated and could explain its beneficial action in both the reduction of osteolysis and prevention of metastasis.

Cerivastatin, an inhibitor of HMG-CoA reductase, inhibits the signaling pathways involved in the invasiveness and metastatic properties of highly invasive breast cancer cell lines: an in vitro study.

Cerivastatin is used in the treatment of hypercholesterolemia to inhibit 3-hydroxy 3-methylglutaryl coenzyme A reductase and thus prevent the synthesis of cholesterol precursors, such as farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), responsible, respectively, for translocation of Ras and Rho to the cell membrane, a step required for their cell signaling, leading to cell proliferation and migration. Recently, it has been suggested that non lipid-related effects of statins could play a beneficial role in cancer therapy. In this study, we have investigated the mechanisms by which statins inhibit cancer and the types of cancers which could benefit from this therapy. In MDA-MB-231 cells, an aggressive breast cancer cell line with spontaneous activation of Ras and NFkappaB and overexpression of RhoA, cerivastatin induced inhibition of both cell proliferation and invasion through Matrigel. This anti-proliferative effect was related to G(1)/S arrest due to an increase in p21(Waf1/Cip1). The anti-invasive effect was observed from 18 h and could be explained by RhoA delocalization from the cell membrane, resulting in disorganization of the actin fibers and disappearance of focal adhesion sites. The importance of RhoA inactivation in both these inhibitory effects was proved by their reversion by GGPP but not by FPP. Moreover, cerivastatin was also shown to induce inactivation of NFkappaB, in a RhoA inhibition-dependent manner, resulting in a decrease in urokinase and metalloproteinase-9 expression, two proteases involved in cell migration. The participation of Ras inactivation is considered a subsidiary mechanism for the effects of cerivastatin, as they were not rescued by FPP. Prolonged treatment of MDA-MB-231 cells with high doses of cerivastatin induced a loss of cell attachment. Interestingly, the effect of cerivastatin was considerably lower on poorly invasive MCF-7 cells. In conclusion, our results suggest that cerivastatin inhibits cell signaling pathways involved in the invasiveness and metastatic properties of highly invasive cancers.

Cooperation between monocytes and breast cancer cells promotes factors involved in cancer aggressiveness.

In breast cancers, clinical symptoms of inflammation localised around the tumour at the time of diagnosis have been considered to have poor prognosis significance. In this study, the biological mechanisms responsible for the deleterious action of monocytes in cancer were investigated. The incubation of the breast-cancer-derived MDA-MB231 cells with monocytes resulted in an increase in factors involved in cell invasion (i.e. both cancer cells and monocytes-associated urokinase and Tissue Factor, and PAI-1 and MMP-9 secretion). Moreover, the functions of monocytes were also modified. Incubation of monocytes with MDA-MB231 cancer cells resulted in a downregulation in the secretion of the antiproliferative cytokine Oncostatin M, while the apoptotic factor TNF alpha was dramatically increased. However, MDA-MB231 cancer cells have been shown to be resistant towards the apoptotic action of TNF alpha. These findings demonstrate that incubation of MDA-MB231 cancer cells with monocytes induced a crosstalk, which resulted in an increased expression of factors involved in cancer cell invasiveness and in a modification of monocytes function against cancer cells, while inflammatory effects were increased.