PSA and androgen-related gene (AR, CYP17, and CYP19) polymorphisms and the risk of adenocarcinoma at prostate biopsy

The aim of the present study was to examine the impact of polymorphisms in prostate-specific antigen (PSA) and androgen-related genes (AR, CYP17, and CYP19) on prostate cancer (PCa) risk in selected high-risk patients who underwent prostate biopsy. Blood samples and prostate tissues were obtained for DNA analysis. Single-nucleotide polymorphisms in the 50-untranslated regions (UTRs) of the PSA (substitution A > G at position -158) and CYP17 (substitution T > C at 50-UTR) genes were detected by polymerase chain reaction (PCR)-restriction fragment length polymorphism assays. The CAG and TTTA repeats in the AR and CYP19 genes, respectively, were genotyped by PCR-based GeneScan analysis. Patients with the GG genotype of the PSA gene had a higher risk of PCa than those with the AG or AA genotype (OR = 3.79, p = 0.00138). The AA genotype was associated with lower PSA levels (6.44 +/- 1.64 ng/mL) compared with genotypes having at least one G allele (10.44 +/- 10.06 ng/mL) (p = 0.0687, 95% CI - 0.3146 to 8.315, unpaired t-test). The multivariate analysis confirmed the association between PSA levels and PSA genotypes (AA vs. AG+GG; chi(2) = 0.0482) and CYP19 (short alleles homozygous vs. at least one long allele; chi(2) = 0.0110) genotypes. Genetic instability at the AR locus leading to somatic mosaicism was detected in one PCa patient by comparing the length of AR CAG repeats in matched peripheral blood and prostate biopsy cores. Taken together, these findings suggest that the PSA genotype should be a clinically relevant biomarker to predict the PCa risk.

Lethal weapons : novel approaches for receptor-targeted cancer cell elimination

The currently used forms of cancer therapy are associated with drug resistance and toxicity to healthy tissues. Thus, more efficient methods are needed for cancer-specific induction of growth arrest and programmed cell death, also known as apoptosis. Therapeutic forms of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) are investigated in clinical trials due to the capability of TRAIL to trigger apoptosis specifically in cancer cells by activation of cell surface death receptors. Many tumors, however, have acquired resistance to TRAIL-induced apoptosis and sensitizing drugs for combinatorial treatments are, therefore, in high demand. This study demonstrates that lignans, natural polyphenols enriched in seeds and cereal, have a remarkable sensitizing effect on TRAIL-induced cell death at non-toxic lignan concentrations. In TRAIL-resistant and androgen-dependent prostate cancer cells we observe that lignans repress receptor tyrosine kinase (RTK) activity and downregulate cell survival signaling via the Akt pathway, which leads to increased TRAIL sensitivity. A structure-activity relationship analysis reveals that the γ-butyrolactone ring of the dibenzylbutyrolactone lignans is essential for the rapidly reversible TRAIL-sensitizing activity of these compounds. Furthermore, the lignan nortrachelogenin (NTG) is identified as the most efficient of the 27 tested lignans and norlignans in sensitization of androgen-deprived prostate cancer cells to TRAIL-induced apoptosis. While this combinatorial anticancer approach may leave normal cells unharmed, several efficient cancer drugs are too toxic, insoluble or unstable to be used in systemic therapy. To enable use of such drugs and to protect normal cells from cytotoxic effects, cancer-targeted drug delivery vehicles of nanometer scale have recently been generated. The newly developed nanoparticle system that we tested in vitro for cancer cell targeting combines the efficient drug-loading capacity of mesoporous silica to the versatile particle surface functionalization of hyperbranched poly(ethylene imine), PEI. The mesoporous hybrid silica nanoparticles (MSNs) were functionalized with folic acid to promote targeted internalization by folate receptor overexpressing cancer cells. The presented results demonstrate that the developed carrier system can be employed in vitro for cancer selective delivery of adsorbed or covalently conjugated molecules and furthermore, for selective induction of apoptotic cell death in folate receptor expressing cancer cells. The tested carrier system displays potential for simultaneous delivery of several anticancer agents specifically to cancer cells also in vivo.

Biomarqueurs pronostiques dans le cancer de la prostate : mieux prédire pour mieux traiter

Le cancer de la prostate (CP) est le cancer le plus fréquemment diagnostiqué en Amérique du Nord et est au troisième rang en termes de létalité chez les hommes. Suite aux traitements de première ligne, 20 à 30% des patients diagnostiqués avec un cancer localisé auront une récidive biochimique de la maladie. La déplétion androgénique mène fréquemment au développement du stade de résistance à la castration (RC). Ce dernier est associé avec une augmentation de la morbidité (métastases osseuses) et de la mortalité avec une survie moyenne inférieure à deux ans. L’évolution du CP est très hétérogène dans la population et il n’existe actuellement aucun biomarqueur pronostique permettant d’identifier les patients à risque de récurrence biochimique, de métastases osseuses et de développement d’une résistance à la castration. De nombreuses études ont démontré que les cytokines inflammatoires IL-6 et IL-8 jouent un rôle dans la pathogénèse du CP, notamment dans le développement de la résistance à la castration. Par ailleurs, les niveaux sériques élevés de ces cytokines ont été associés à un mauvais pronostic. Précédemment, notre laboratoire a démontré in vitro que la protéine IKKε entraîne une augmentation de la sécrétion de ces cytokines dans les cellules du CP et qu’elle est exprimée davantage dans les tissus de cancers plus avancés. Le premier objectif du présent mémoire fut d’évaluer dans des tissus humains la corrélation d’IKKε, IL-6 et IL-8 avec des paramètres cliniques. Nos résultats soulignent le potentiel d’IKKε comme biomarqueur tissulaire pronostique de récurrence biochimique et de métastases osseuses. Nous n’avons trouvé aucune association entre IL-6/IL-8 et les paramètres cliniques inclus dans l’étude. Le second objectif de ce projet fut d’évaluer la coexpression de ces trois molécules dans l’épithélium du CP. Nos résultats confirment les observations in vitro en mettant en évidence une forte association entre l’expression d’IKKε, IL-6 et IL-8. Le troisième objectif fut d’évaluer la relation entre les niveaux sériques et tissulaires d’IL-6 et d’IL-8. Aucune relation significative n’a été établie, suggérant que les cytokines sériques ne sont pas uniquement d’origine prostatique. En conclusion, mon projet de maîtrise aura permis de préciser le potentiel d’IKKε comme biomarqueur tissulaire pronostique et de valider pour la première fois dans des tissus humains sa co-expression avec les cytokines IL-6 et IL-8, dont le rôle dans la pathogénèse de la maladie est bien établi. Une étude plus exhaustive des voies de signalisation d’IKKε reste d’intérêt pour élucider notamment les mécanismes par lesquels IKKε stimule la production de cytokines et par quels moyens cette protéine pourrait être impliquée dans le développement d’un état résistant à la castration.

Tissue changes in senescent gerbil prostate after hormone deprivation leads to acquisition of androgen insensitivity

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Biomarqueurs pronostiques dans le cancer de la prostate : mieux prédire pour mieux traiter

Le cancer de la prostate (CP) est le cancer le plus fréquemment diagnostiqué en Amérique du Nord et est au troisième rang en termes de létalité chez les hommes. Suite aux traitements de première ligne, 20 à 30% des patients diagnostiqués avec un cancer localisé auront une récidive biochimique de la maladie. La déplétion androgénique mène fréquemment au développement du stade de résistance à la castration (RC). Ce dernier est associé avec une augmentation de la morbidité (métastases osseuses) et de la mortalité avec une survie moyenne inférieure à deux ans. L’évolution du CP est très hétérogène dans la population et il n’existe actuellement aucun biomarqueur pronostique permettant d’identifier les patients à risque de récurrence biochimique, de métastases osseuses et de développement d’une résistance à la castration. De nombreuses études ont démontré que les cytokines inflammatoires IL-6 et IL-8 jouent un rôle dans la pathogénèse du CP, notamment dans le développement de la résistance à la castration. Par ailleurs, les niveaux sériques élevés de ces cytokines ont été associés à un mauvais pronostic. Précédemment, notre laboratoire a démontré in vitro que la protéine IKKε entraîne une augmentation de la sécrétion de ces cytokines dans les cellules du CP et qu’elle est exprimée davantage dans les tissus de cancers plus avancés. Le premier objectif du présent mémoire fut d’évaluer dans des tissus humains la corrélation d’IKKε, IL-6 et IL-8 avec des paramètres cliniques. Nos résultats soulignent le potentiel d’IKKε comme biomarqueur tissulaire pronostique de récurrence biochimique et de métastases osseuses. Nous n’avons trouvé aucune association entre IL-6/IL-8 et les paramètres cliniques inclus dans l’étude. Le second objectif de ce projet fut d’évaluer la coexpression de ces trois molécules dans l’épithélium du CP. Nos résultats confirment les observations in vitro en mettant en évidence une forte association entre l’expression d’IKKε, IL-6 et IL-8. Le troisième objectif fut d’évaluer la relation entre les niveaux sériques et tissulaires d’IL-6 et d’IL-8. Aucune relation significative n’a été établie, suggérant que les cytokines sériques ne sont pas uniquement d’origine prostatique. En conclusion, mon projet de maîtrise aura permis de préciser le potentiel d’IKKε comme biomarqueur tissulaire pronostique et de valider pour la première fois dans des tissus humains sa co-expression avec les cytokines IL-6 et IL-8, dont le rôle dans la pathogénèse de la maladie est bien établi. Une étude plus exhaustive des voies de signalisation d’IKKε reste d’intérêt pour élucider notamment les mécanismes par lesquels IKKε stimule la production de cytokines et par quels moyens cette protéine pourrait être impliquée dans le développement d’un état résistant à la castration.

Adipocyte Secreted Factors Enhance Aggressiveness of Prostate Carcinoma Cells

Obesity has been associated with increased incidence and risk of mortality of prostate cancer. One of the proposed mechanisms underlying this risk association is the change in adipokines expression that could promote the development and progression of the prostate tumor cells. The main goal of this study was to evaluate the effect of preadipocyte and adipocyte secretome in the proliferation, migration and invasion of androgen independent prostate carcinoma cells (RM1) and to assess cell proliferation in the presence of the adiposity signals leptin and insulin. RM1 cells were co-cultured in with preadipocytes, adipocytes or cultured in their respective conditioned medium. Cell proliferation was assessed by flow cytometry and XTT viability test. Cell migration was evaluated using a wound healing injury assay of RM1 cells cultured with conditioned media. Cellular invasion of RM1 cells co-cultured with adipocytes and preadipocytes was assessed using matrigel membranes. Preadipocyte conditioned medium was associated with a small increase in RM1 proliferation, while adipocytes conditioned media significantly increased RM1 cell proliferation (p<0.01). Adipocytes also significantly increased the RM1 cells proliferation in co-culture (p <0.01). Cell migration was higher in RM1 cells cultured with preadipocyte and adipocyte conditioned medium. RM1 cell invasion was significantly increased after co-culture with preadipocytes and adipocytes (p <0.05). Insulin also increased significantly the cell proliferation in contrast to leptin, which showed no effect. In conclusion, prostate carcinoma cells seem to be influenced by factors secreted by adipocytes that are able to increase their ability to proliferate, migrate and invade.

Development of a T Cell Receptor Targeting an HLA-A*0201 Restricted Epitope from the Cancer-Testis Antigen SSX2 for Adoptive Immunotherapy of Cancer.

The clinical success of adoptive immunotherapy of cancer relies on the selection of target antigens that are highly expressed in tumor cells but absent in essential normal tissues. A group of genes that encode the cancer/testis or cancer germline antigens have been proposed as ideal targets for immunotherapy due to their high expression in multiple cancer types and their restricted expression in immunoprivileged normal tissues. In the present work we report the isolation and characterization of human T cell receptors (TCRs) with specificity for synovial sarcoma X breakpoint 2 (SSX2), a cancer/testis antigen expressed in melanoma, prostate cancer, lymphoma, multiple myeloma and pancreatic cancer, among other tumors. We isolated seven HLA-A2 restricted T cell receptors from natural T cell clones derived from tumor-infiltrated lymph nodes of two SSX2-seropositive melanoma patients, and selected four TCRs for cloning into retroviral vectors. Peripheral blood lymphocytes (PBL) transduced with three of four SSX2 TCRs showed SSX241-49 (KASEKIFYV) peptide specific reactivity, tumor cell recognition and tetramer binding. One of these, TCR-5, exhibited tetramer binding in both CD4 and CD8 cells and was selected for further studies. Antigen-specific and HLA-A*0201-restricted interferon-γ release, cell lysis and lymphocyte proliferation was observed following culture of TCR engineered human PBL with relevant tumor cell lines. Codon optimization was found to increase TCR-5 expression in transduced T cells, and this construct has been selected for development of clinical grade viral vector producing cells. The tumor-specific pattern of expression of SSX2, along with the potent and selective activity of TCR-5, makes this TCR an attractive candidate for potential TCR gene therapy to treat multiple cancer histologies.

Functional chromatin features are associated with structural mutations in cancer.

BACKGROUND: Structural mutations (SMs) play a major role in cancer development. In some cancers, such as breast and ovarian, DNA double-strand breaks (DSBs) occur more frequently in transcribed regions, while in other cancer types such as prostate, there is a consistent depletion of breakpoints in transcribed regions. Despite such regularity, little is understood about the mechanisms driving these effects. A few works have suggested that protein binding may be relevant, e.g. in studies of androgen receptor binding and active chromatin in specific cell types. We hypothesized that this behavior might be general, i.e. that correlation between protein-DNA binding (and open chromatin) and breakpoint locations is common across divergent cancers. RESULTS: We investigated this hypothesis by comprehensively analyzing the relationship among 457 ENCODE protein binding ChIP-seq experiments, 125 DnaseI and 24 FAIRE experiments, and 14,600 SMs from 8 diverse cancer datasets covering 147 samples. In most cancers, including breast and ovarian, we found enrichment of protein binding and open chromatin in the vicinity of SM breakpoints at distances up to 200 kb. Furthermore, for all cancer types we observed an enhanced enrichment in regions distant from genes when compared to regions proximal to genes, suggesting that the SM-induction mechanism is independent from the bias of DSBs to occur near transcribed regions. We also observed a stronger effect for sites with more than one protein bound. CONCLUSIONS: Protein binding and open chromatin state are associated with nearby SM breakpoints in many cancer datasets. These observations suggest a consistent mechanism underlying SM locations across different cancers.

Role and Function of c-Jun Protein Complex in Cancer Cell Behavior

Transcription factors play a crucial role in the regulation of cell behavior by modulating gene expression profiles. Previous studies have described a dual role for the AP-1 family transcription factor c-Jun in the regulation of cellular fate. In various cell types weak and transient activations of c-Jun N-terminal kinase (JNK) and c-Jun appear to contribute to proliferation and survival, whereas strong and prolonged activation of JNK and c-Jun result in apoptosis. These opposite roles played by c-Jun are cell type specific and the molecular mechanisms defining these antonymous c-Jun-mediated responses remain incompletely understood. c-Jun activity in transformed cells is regulated by signalling cascades downstream of oncoproteins such as Ras and Raf. In addition, the pro-proliferative role and the survival promoting function for c-Jun has been described in various cancer models. Furthermore, c-Jun was described to be overexpressed in different cancer types. However, the molecular mechanisms by which c-Jun exerts these oncogenic functions are not all clearly established. Therefore it is of primary interest to further identify molecular mechanisms and functions for c-Jun in cancer. Regulation of gene expression is tightly dependent on accurate protein-protein interactions. Therefore, co-factors for c-Jun may define the functions for c-Jun in cancer. Identification of protein-protein interactions promoting cancer may provide novel possibilities for cancer treatment. In this study, we show that DNA topoisomerase I (TopoI) is a transcriptional co-factor for c-Jun. Moreover, c-Jun and TopoI together promote expression of epidermal growth factor receptor (EGFR) in cancer cells. We also show that the clinically used TopoI inhibitor topotecan reduces EGFR expression. Importantly, the effect of TopoI on EGFR transcription was shown to depend on c-Jun as Jun-/- cells or cells treated with JNK inhibitor SP600125 are resistant to topotecan treatment both in regulation of EGFR expression and cell proliferation. Moreover, c-Jun regulates the nucleolar localization and the function of the ribonucleic acid (RNA) helicase DDX21, a previously identified member of c-Jun protein complex. In addition, c-Jun stimulates rRNA processing by supporting DDX21 rRNA binding. Finally, this study characterizes a DDX21 dependent expression of cyclin dependent kinase (Cdk) 6, a correlation of DDX21 expression with prostate cancer progression and a substrate binding dependency of DDX21 nucleolar localization in prostate cancer cells. Taken together, the results of this study validate the c-Jun-TopoI interaction and precise the c-Jun-DDX21 interaction. Moreover, these results show the importance for protein-protein interaction in the regulation of their cellular functions in cancer cell behavior. Finally, the results presented here disclose new exciting therapeutic opportunities for cancer treatment.

Fibroblast Growth Factor 8 and its Receptors in The Growth and Angiogenesis of Experimental Breast Cancer . With Special Reference to Thrombospondin-1 as a Novel Target Gene for FGF-8

The growth of breast cancer is regulated by hormones and growth factors. Recently, aberrant fibroblast growth factor (FGF) signalling has been strongly implicated in promoting the progression of breast cancer and is thought to have a role in the development of endocrine resistant disease. FGFs mediate their auto- and paracrine signals through binding to FGF receptors 1-4 (FGFR1-4) and their isoforms. Specific targets of FGFs in breast cancer cells and the differential role of FGFRs, however, are poorly described. FGF-8 is expressed at elevated levels in breast cancer, and it has been shown to act as an angiogenic, growth promoting factor in experimental models of breast cancer. Furthermore, it plays an important role in mediating androgen effects in prostate cancer and in some breast cancer cell lines. We aimed to study testosterone (Te) and FGF-8 regulated genes in Shionogi 115 (S115) breast cancer cells, characterise FGF-8 activated intracellular signalling pathways and clarify the role of FGFR1, -2 and -3 in these cells. Thrombospondin-1 (TSP-1), an endogenous inhibitor of angiogenesis, was recognised as a Te and FGF-8 regulated gene. Te repression of TSP-1 was androgen receptor (AR)-dependent. It required de novo protein synthesis, but it was independent of FGF-8 expression. FGF-8, in turn, downregulated TSP-1 transcription by activating the ERK and PI3K pathways, and the effect could be reversed by specific kinase inhibitors. Differential FGFR1-3 action was studied by silencing each receptor by shRNA expression in S115 cells. FGFR1 expression was a prerequisite for the growth of S115 tumours, whereas FGFR2 expression alone was not able to promote tumour growth. High FGFR1 expression led to a growth advantage that was associated with strong ERK activation, increased angiogenesis and reduced apoptosis, and all of these effects could be reversed by an FGFR inhibitor. Taken together, the results of this thesis show that FGF-8 and FGFRs contribute strongly to the regulation of the growth and angiogenesis of experimental breast cancer and support the evidence for FGF-FGFR signalling as one of the major players in breast cancers.

To divide or differentiate? : nestin-mediated regulation of Cdk5 in cell fate decisions

The molecular functions of the non-cell cycle-related Cyclin-dependent kinase 5 (Cdk5) have been of primary interest within the neuroscience field, but novel undertakings are constantly emerging for the kinase in tissue homeostasis, as well as in diseases such as diabetes and cancer. Although Cdk5 activation is predominantly regulated by specific non-cyclin activator protein binding, additional mechanisms have proved to orchestrate Cdk5 signaling in cells. For example, the interaction between the intermediate filament protein nestin and Cdk5 has been proposed to determine cellular fate during neuronal apoptosis through nestin-dependent adjustment of the sensitive balance and turnover of Cdk5 activators. While nestin constitutes a crucial regulatory scaffold for appropriate Cdk5 activation in apoptosis, Cdk5 itself phosphorylates nestin with the consequence of filament reorganization in both neuronal progenitors and differentiating muscle cells. Interestingly, the two proteins are often found coexpressed in various tissues and cell types, proposing that nestin-mediated scaffolding of Cdk5 and its activators may be applicable to other tissue systems as well. In the literature, the molecular functions of nestin have remained in the shade, as it is mostly exploited as a marker protein for progenitor cells. In light of these studies, the aim of this thesis was to assess the importance of the nestin scaffold in regulation of Cdk5 actions in cell fate decisions. This thesis can be subdivided into two major projects: one that studied the nature of the Cdk5-nestin interplay in muscle, and one that assessed their role in prostate cancer. During differentiation of a myoblast cell line, the filament formation properties of nestin was found to be crucial in directing Cdk5 activity, with direct consequences on the process of differentiation. Also the genetic knockout of nestin was found to influence Cdk5 activity, although differentiation per se was not affected. Instead, the genetic ablation of nestin had broad consequences on muscle homeostasis and regeneration. While the nestin-mediated regulation of Cdk5 in muscle was found to act in multiple ways, the connection remained more elusive in cancer models. Cdk5 was, however, established as a significant determinant of prostate cancer proliferation; a behavior uncharacteristic for this differentiation-associated kinase. Through complex and simultaneous regulation of two major prostate cancer pathways, Cdk5 was placed upstream of both Akt kinase and the androgen receptor. Its action on proliferation was nonetheless mainly exerted through the Akt signaling pathway in various cancer models. In summary, this thesis contributed to the knowledge of Cdk5 regulation and functions in two atypical settings; proliferation (in a cancer framework) and muscle differentiation, which is a poorly understood model system in the Cdk5 field. This balance between proliferation and differentiation implemented by Cdk5 is ultimately regulated (where present) by the dynamics of the cytoskeletal nestin scaffold.

Construction of a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen and mouse 4-1BBL genes and its effect on dendritic cells

Our aim was to construct a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen (tPSMA) and mouse 4-1BBL genes and to determine its effect on dendritic cells (DCs) generated from bone marrow suspensions harvested from C57BL/6 mice for which the effect of 4-1BBL on DCs is not clear, especially during DCs processing tumor-associated antigen. Replication deficient adenovirus AdMaxTM Expression System was used to construct recombinant adenovirus Ad-tPSMA-internal ribosome entry site-mouse 4-1BBL (Ad-tPSMA-IRES-m4-1BBL) and Ad-enhanced green fluorescent protein. Day 7 proliferating DC aggregates generated from C57BL/6 mice were collected as immature DCs and further mature DCs were obtained by lipopolysaccharide activated immature DCs. After DCs were exposed to the recombinant adenovirus with 250 multiplicity of infection, the expression of tPSMA and m4-1BBL proteins were detected by Western blot, and the apoptosis and phenotype of DCs were analyzed by flow cytometry. Cytokines (IL-6 and IL-12) in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Proliferation of T cells was detected by allogeneic mixed lymphocyte reactions. The tPSMA and m4-1BBL proteins were expressed correctly. The apoptosis rate of DCs transfected with Ad-tPSMA-IRES-m4-1BBL was 14.6%, lower than that of control DCs. The expression of co-stimulatory molecules [CD80 (81.6 ± 5.4%) and CD86 (80.13 ± 2.81%)] up-regulated in Ad-tPSMA-IRES-m4-1BBL-pulsed DCs, and the level of IL-6 (3960.2 ± 50.54 pg/mL) and IL-12 (249.57 ± 12.51 pg/mL) production in Ad-tPSMA-IRES-m4-1BBL-transduced DCs were significantly higher (P < 0.05) than those in control DCs. Ad-tPSMA-IRES-m4-1BBL induced higher T-cell proliferation (OD450 = 0.614 ± 0.018), indicating that this recombinant adenovirus can effectively enhance the activity of DCs.

Rôle de GPR40 dans la survie et la prolifération cellulaires induites par l’oléate dans les cellules de cancer du sein MDA-MB-231 et de cancer de la prostate DU145

La relation entre l’obésité et le cancer, bien qu’établie par des études épidémiologiques, est peu connue. Pourtant, environ 25 % des cancers pourraient y être attribuables. Parmi les cancers reliés à l’obésité, les cancers du côlon, du sein chez les femmes ménopausées et de la prostate sont les plus fréquents. Des études sur modèles animaux ont suggéré une association positive entre une diète riche en gras et le développement du cancer mammaire et de la prostate. Nous avons étudié les mécanismes moléculaires par lesquels les acides gras influencent le devenir de lignées de cellules cancéreuses du sein et de la prostate. Ces travaux ont montré que les acides gras insaturés, dont l’oléate, induisent la prolifération cellulaire tandis que les acides gras saturés, dont le palmitate, diminuent la prolifération. Un traitement à l’oléate stimule la formation de gouttelettes lipidiques dans le cytoplasme des cellules de cancer du sein MDA-MB-231 et de la prostate DU145 alors qu’un traitement au palmitate entraîne l’apoptose. Le mécanisme d’action de l’oléate sur la prolifération a été étudié de façon plus approfondie. L’utilisation d’inhibiteurs pharmacologiques nous a permis de déterminer que l’effet prolifératif de l’oléate implique la voie PI3K/Akt, la voie ERK1/2 et l’activation d’un ou de plusieurs récepteur(s) couplé(s) aux protéines G (GPCR). L’oléate induit la phosphorylation rapide des protéines Akt et ERK1/2 dans les cellules de cancer du sein MDA-MB-231 et de la prostate DU145. Au cours des dernières années, deux GPCRs ont été identifiés comme étant activables par des acides gras à moyennes et à longues chaînes, GPR40 et GPR120. GPR40 étant exprimé dans plusieurs lignées cellulaires de cancer du sein et de la prostate contrairement à l’expression de GPR120 qui était inexistante dans la plupart des lignées, nous avons étudié l’implication de GPR40 dans l’effet prolifératif de l’oléate. Ces deux récepteurs n’étant pas exprimés dans les cellules épithéliales mammaires humaines en culture primaire, ces cellules ne répondent pas aux effets de l’oléate sur la prolifération et l’activation des voies de signalisation. L’activation des voies Akt et ERK1/2 par l’oléate dans les cellules MDA-MB-231 et DU145 est potentialisée par la surexpression du récepteur GPR40 et inhibée par l’utilisation d’un siRNA dirigé contre ce récepteur. Cependant, la prolifération induite par l’oléate ne semble pas affectée par la présence d’un siRNA dirigé contre GPR40. L’oléate étant un acide gras, il est capable d’entrer librement dans les cellules et une partie de ses effets sur la prolifération pourrait être attribuée à sa métabolisation. Un agoniste de GPR40, le GW9508, est en mesure d’activer GPR40 sans toutefois entrer dans les cellules ni activer le métabolisme de l’oléate. Le GW9508 stimule la phosphorylation des protéines Akt et ERK1/2 dans les cellules du cancer du sein MDA-MB-231 et de la prostate DU145, mais il n’est pas en mesure d’induire la prolifération cellulaire comme le fait l’oléate. Ces résultats nous permettent de mieux comprendre le mécanisme d’action de l’oléate sur les cellules de cancer du sein et de la prostate. L’oléate induit la signalisation de GPR40 qui est impliquée dans l’activation rapide des voies de signalisation Akt et ERK1/2. De son côté, l’effet prolifératif induit par l’oléate s’effectue par un mécanisme GPR40-indépendant, possiblement lié au métabolisme de l’oléate.

Genotyping of AR and PSA polymorphisms in a patient with Klinefelter syndrome, non-Hodgkin lymphoma, and adenocarcinoma of the prostate

It has been hypothesized that the AR (androgen receptor) gene binds the two PSA (prostate-specific antigen) alleles with differing affinities and may differentially influence prostate cancer risk. In this article, we report a case of adenocarcinoma of the prostate in a 56-year-old man with Klinefelter syndrome (47,XXY) and non-Hodgkin lymphoma, as well as the AR and PSA genotype. AR and PSA gene polymorphisms were analyzed by polymerase chain reaction-based methods using DNA from peripheral white blood cells and the prostate cancer. We determined the methylation status of the AR gene on the X chromosome. The patient presents with the AG genotype for the ARE-I (androgen response element) region of the PSA gene. We detect the presence of two short AR alleles with 19 and 11CAG repeats each. Unmethylated alleles were demonstrated for both. The shorter allele was inactive in more than 60% of total DNA in both control blood and prostate cancer cells. The presence of short AR alleles and the G allele of the PSA gene may contribute to the development of prostate cancer in a 47,XXY patient. (C) 2004 Elsevier B.V. All rights reserved.

PSA and androgen-related gene (AR, CYP17, and CYP19) polymorphisms and the risk of adenocarcinoma at prostate biopsy

The aim of the present study was to examine the impact of polymorphisms in prostate-specific antigen (PSA) and androgen-related genes (AR, CYP17, and CYP19) on prostate cancer (PCa) risk in selected high-risk patients who underwent prostate biopsy. Blood samples and prostate tissues were obtained for DNA analysis. Single-nucleotide polymorphisms in the 50-untranslated regions (UTRs) of the PSA (substitution A > G at position -158) and CYP17 (substitution T > C at 50-UTR) genes were detected by polymerase chain reaction (PCR)-restriction fragment length polymorphism assays. The CAG and TTTA repeats in the AR and CYP19 genes, respectively, were genotyped by PCR-based GeneScan analysis. Patients with the GG genotype of the PSA gene had a higher risk of PCa than those with the AG or AA genotype (OR = 3.79, p = 0.00138). The AA genotype was associated with lower PSA levels (6.44 +/- 1.64 ng/mL) compared with genotypes having at least one G allele (10.44 +/- 10.06 ng/mL) (p = 0.0687, 95% CI - 0.3146 to 8.315, unpaired t-test). The multivariate analysis confirmed the association between PSA levels and PSA genotypes (AA vs. AG+GG; chi(2) = 0.0482) and CYP19 (short alleles homozygous vs. at least one long allele; chi(2) = 0.0110) genotypes. Genetic instability at the AR locus leading to somatic mosaicism was detected in one PCa patient by comparing the length of AR CAG repeats in matched peripheral blood and prostate biopsy cores. Taken together, these findings suggest that the PSA genotype should be a clinically relevant biomarker to predict the PCa risk.