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1.
Pharmaceutics ; 14(5)2022 May 05.
Article in English | MEDLINE | ID: mdl-35631574

ABSTRACT

Despite the development of new therapeutic strategies, cancer remains one of the leading causes of mortality worldwide. One of the current major challenges is the resistance of cancers to chemotherapy treatments inducing metastases and relapse of the tumor. The Hedgehog receptor Patched (Ptch1) is overexpressed in many types of cancers. We showed that Ptch1 contributes to the efflux of doxorubicin and plays an important role in the resistance to chemotherapy in adrenocortical carcinoma (ACC), a rare cancer which presents strong resistance to the standard of care chemotherapy treatment. In the present study, we isolated and characterized a subpopulation of the ACC cell line H295R in which Ptch1 is overexpressed and more present at the cell surface. This cell subpopulation is more resistant to doxorubicin, grows as spheroids, and has a greater capability of clonogenicity, migration, and invasion than the parental cells. Xenograft experiments performed in mice and in ovo showed that this cell subpopulation is more tumorigenic and metastatic than the parental cells. These results suggest that this cell subpopulation has cancer stem-like or persistent cell properties which were strengthened by RNA-seq. If present in tumors from ACC patients, these cells could be responsible for therapy resistance, relapse, and metastases.

2.
Cancer Res ; 81(13): 3480-3494, 2021 07 01.
Article in English | MEDLINE | ID: mdl-34127497

ABSTRACT

Succinate dehydrogenase is a key enzyme in the tricarboxylic acid cycle and the electron transport chain. All four subunits of succinate dehydrogenase are tumor suppressor genes predisposing to paraganglioma, but only mutations in the SDHB subunit are associated with increased risk of metastasis. Here we generated an Sdhd knockout chromaffin cell line and compared it with Sdhb-deficient cells. Both cell types exhibited similar SDH loss of function, metabolic adaptation, and succinate accumulation. In contrast, Sdhb-/- cells showed hallmarks of mesenchymal transition associated with increased DNA hypermethylation and a stronger pseudo-hypoxic phenotype compared with Sdhd-/- cells. Loss of SDHB specifically led to increased oxidative stress associated with dysregulated iron and copper homeostasis in the absence of NRF2 activation. High-dose ascorbate exacerbated the increase in mitochondrial reactive oxygen species, leading to cell death in Sdhb-/- cells. These data establish a mechanism linking oxidative stress to iron homeostasis that specifically occurs in Sdhb-deficient cells and may promote metastasis. They also highlight high-dose ascorbate as a promising therapeutic strategy for SDHB-related cancers. SIGNIFICANCE: Loss of different succinate dehydrogenase subunits can lead to different cell and tumor phenotypes, linking stronger 2-OG-dependent dioxygenases inhibition, iron overload, and ROS accumulation following SDHB mutation.


Subject(s)
Ascorbic Acid/pharmacology , Homeostasis , Iron/metabolism , Mutation , Oxidative Stress , Succinate Dehydrogenase/physiology , Animals , Antioxidants/pharmacology , Dioxygenases/antagonists & inhibitors , Female , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitochondria/metabolism , Mitochondria/pathology , Phenotype , Reactive Oxygen Species
3.
Cell Rep ; 30(13): 4551-4566.e7, 2020 03 31.
Article in English | MEDLINE | ID: mdl-32234487

ABSTRACT

Loss-of-function mutations in the SDHB subunit of succinate dehydrogenase predispose patients to aggressive tumors characterized by pseudohypoxic and hypermethylator phenotypes. The mechanisms leading to DNA hypermethylation and its contribution to SDH-deficient cancers remain undemonstrated. We examine the genome-wide distribution of 5-methylcytosine and 5-hydroxymethylcytosine and their correlation with RNA expression in SDHB-deficient tumors and murine Sdhb-/- cells. We report that DNA hypermethylation results from TET inhibition. Although it preferentially affects PRC2 targets and known developmental genes, PRC2 activity does not contribute to the DNA hypermethylator phenotype. We also prove, in vitro and in vivo, that TET silencing, although recapitulating the methylation profile of Sdhb-/- cells, is not sufficient to drive their EMT-like phenotype, which requires additional HIF2α activation. Altogether, our findings reveal synergistic roles of TET repression and pseudohypoxia in the acquisition of metastatic traits, providing a rationale for targeting HIF2α and DNA methylation in SDH-associated malignancies.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , DNA Methylation/genetics , DNA-Binding Proteins/metabolism , Mesoderm/metabolism , Proto-Oncogene Proteins/metabolism , Succinate Dehydrogenase/genetics , 5-Methylcytosine/analogs & derivatives , 5-Methylcytosine/metabolism , Adult , Aged , Animals , Cell Hypoxia , Cell Line , Cell Line, Tumor , Dioxygenases , Female , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Genome, Human , Humans , Male , Mice, Nude , Middle Aged , Mutation/genetics , Neoplasm Metastasis , Phenotype , Polycomb Repressive Complex 2/metabolism , Succinate Dehydrogenase/deficiency
4.
Cancer Res ; 78(8): 1914-1922, 2018 04 15.
Article in English | MEDLINE | ID: mdl-29431636

ABSTRACT

Comprehensive genetic analyses have identified germline SDHB and FH gene mutations as predominant causes of metastatic paraganglioma and pheochromocytoma. However, some suspicious cases remain unexplained. In this study, we performed whole-exome sequencing of a paraganglioma exhibiting an SDHx-like molecular profile in the absence of SDHx or FH mutations and identified a germline mutation in the SLC25A11 gene, which encodes the mitochondrial 2-oxoglutarate/malate carrier. Germline SLC25A11 mutations were identified in six other patients, five of whom had metastatic disease. These mutations were associated with loss of heterozygosity, suggesting that SLC25A11 acts as a tumor-suppressor gene. Pseudohypoxic and hypermethylator phenotypes comparable with those described in SDHx- and FH-related tumors were observed both in tumors with mutated SLC25A11 and in Slc25a11Δ/Δ immortalized mouse chromaffin knockout cells generated by CRISPR-Cas9 technology. These data show that SLC25A11 is a novel paraganglioma susceptibility gene for which loss of function correlates with metastatic presentation.Significance: A gene encoding a mitochondrial carrier is implicated in a hereditary cancer predisposition syndrome, expanding the role of mitochondrial dysfunction in paraganglioma. Cancer Res; 78(8); 1914-22. ©2018 AACR.


Subject(s)
Adrenal Gland Neoplasms/genetics , Adrenal Gland Neoplasms/pathology , Genetic Predisposition to Disease , Germ-Line Mutation , Membrane Transport Proteins/genetics , Paraganglioma/secondary , Pheochromocytoma/genetics , Animals , CRISPR-Cas Systems , Cohort Studies , Humans , Loss of Heterozygosity , Mice , Mice, Knockout , Mutation , Neoplasm Metastasis , Paraganglioma/genetics , Phenotype , Pheochromocytoma/secondary
5.
Cell Tissue Res ; 372(2): 379-392, 2018 05.
Article in English | MEDLINE | ID: mdl-29427052

ABSTRACT

Paragangliomas and pheochromocytomas are rare neuroendocrine tumors characterized by a large spectrum of hereditary predisposition. Based on gene expression profiling classification, they can be classically assigned to either a hypoxic/angiogenic cluster (cluster 1 including tumors with mutations in SDHx, VHL and FH genes) or a kinase-signaling cluster (cluster 2 consisting in tumors related to RET, NF1, TMEM127 and MAX genes mutations, as well as most of the sporadic tumors). The past 15 years have seen the emergence of an increasing number of genetically engineered and grafted models to investigate tumorigenesis and develop new therapeutic strategies. Among them, only cluster 2-related predisposed models have been successful but grafted models are however available to study cluster 1-related tumors. In this review, we present an overview of existing rodent models targeting predisposition genes involved or not in human pheochromocytoma/paraganglioma susceptibility and their contribution to the improvement of pheochromocytoma experimental research.


Subject(s)
Adrenal Gland Neoplasms/pathology , Carcinogenesis/pathology , Disease Models, Animal , Pheochromocytoma/pathology , Adrenal Gland Neoplasms/genetics , Animals , Carcinogenesis/genetics , Genetic Predisposition to Disease , Humans , Pheochromocytoma/genetics , Rodentia
6.
Oncotarget ; 8(31): 51050-51057, 2017 Aug 01.
Article in English | MEDLINE | ID: mdl-28881628

ABSTRACT

Adrenocortical carcinoma is a rare neoplasm with a poor prognosis. Very important advances have been made in the identification of the genetic determinants of adrenocortical carcinoma pathogenesis but our understanding is still limited about the mechanisms that determine cancer spread and metastasis. One major problem hindering preclinical experimentation for new therapies for adrenocortical carcinoma is represented by the lack of suitable animal models for metastatic disease. With the aim to overcome these limitations, in this study we tested several protocols in order to establish a mouse xenograft model of metastatic adrenocortical carcinoma. The most efficient method, based upon intrasplenic injection followed by splenectomy, produced metastases with high efficiency, whose development could be followed over time by bioluminescence measurements. We expect that the availability of this model will greatly improve the possibilities for preclinical testing of new treatments for advanced-stage disease.

7.
Sci Signal ; 10(469)2017 Mar 07.
Article in English | MEDLINE | ID: mdl-28270555

ABSTRACT

Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with a dismal prognosis. Genomic studies have enabled progress in our understanding of the molecular bases of ACC, but factors that influence its prognosis are lacking. Amplification of the gene encoding the transcription factor steroidogenic factor-1 (SF-1; also known as NR5A1) is one of the genetic alterations common in ACC. We identified a transcriptional regulatory mechanism involving increased abundance of VAV2, a guanine nucleotide exchange factor for small GTPases that control the cytoskeleton, driven by increased expression of the gene encoding SF-1 in ACC. Manipulating SF-1 and VAV2 abundance in cultured ACC cells revealed that VAV2 was a critical factor for SF-1-induced cytoskeletal remodeling and invasion in culture (Matrigel) and in vivo (chicken chorioallantoic membrane) models. Analysis of ACC patient cohorts indicated that greater VAV2 abundance robustly correlated with poor prognosis in ACC patients. Because VAV2 is a druggable target, our findings suggest that blocking VAV2 may be a new therapeutic approach to inhibit metastatic progression in ACC patients.


Subject(s)
Adrenal Cortex Neoplasms/genetics , Adrenocortical Carcinoma/genetics , Gene Expression Regulation, Neoplastic , Proto-Oncogene Proteins c-vav/genetics , Steroidogenic Factor 1/genetics , Adolescent , Adrenal Cortex Neoplasms/metabolism , Adrenal Cortex Neoplasms/pathology , Adrenocortical Carcinoma/metabolism , Adrenocortical Carcinoma/pathology , Adult , Aged , Aged, 80 and over , Animals , Cell Line, Tumor , Chick Embryo , Chorioallantoic Membrane/metabolism , Cytoskeleton/metabolism , Female , Humans , Male , Middle Aged , Neoplasm Invasiveness , Proto-Oncogene Proteins c-vav/metabolism , Steroidogenic Factor 1/metabolism , Survival Analysis , Young Adult
8.
Clin Cancer Res ; 22(5): 1120-9, 2016 Mar 01.
Article in English | MEDLINE | ID: mdl-26490314

ABSTRACT

PURPOSE: Germline mutations in genes encoding mitochondrial succinate dehydrogenase (SDH) are found in patients with paragangliomas, pheochromocytomas, gastrointestinal stromal tumors, and renal cancers. SDH inactivation leads to a massive accumulation of succinate, acting as an oncometabolite and which levels, assessed on surgically resected tissue are a highly specific biomarker of SDHx-mutated tumors. The aim of this study was to address the feasibility of detecting succinate in vivo by magnetic resonance spectroscopy. EXPERIMENTAL DESIGN: A pulsed proton magnetic resonance spectroscopy ((1)H-MRS) sequence was developed, optimized, and applied to image nude mice grafted with Sdhb(-/-) or wild-type chromaffin cells. The method was then applied to patients with paraganglioma carrying (n = 5) or not (n = 4) an SDHx gene mutation. Following surgery, succinate was measured using gas chromatography/mass spectrometry, and SDH protein expression was assessed by immunohistochemistry in resected tumors. RESULTS: A succinate peak was observed at 2.44 ppm by (1)H-MRS in all Sdhb(-/-)-derived tumors in mice and in all paragangliomas of patients carrying an SDHx gene mutation, but neither in wild-type mouse tumors nor in patients exempt of SDHx mutation. In one patient, (1)H-MRS results led to the identification of an unsuspected SDHA gene mutation. In another case, it helped define the pathogenicity of a variant of unknown significance in the SDHB gene. CONCLUSIONS: Detection of succinate by (1)H-MRS is a highly specific and sensitive hallmark of SDHx mutations. This noninvasive approach is a simple and robust method allowing in vivo detection of the major biomarker of SDHx-mutated tumors.


Subject(s)
Electron Transport Complex II/genetics , Membrane Proteins/genetics , Paraganglioma/genetics , Pheochromocytoma/genetics , Succinate Dehydrogenase/genetics , Animals , Genetic Predisposition to Disease , Germ-Line Mutation/genetics , Humans , Magnetic Resonance Spectroscopy , Male , Mice , Paraganglioma/metabolism , Paraganglioma/pathology , Pheochromocytoma/pathology , Succinic Acid/metabolism , Xenograft Model Antitumor Assays
9.
Mol Cell Endocrinol ; 421: 40-8, 2016 Feb 05.
Article in English | MEDLINE | ID: mdl-26123588

ABSTRACT

Pheochromocytomas and paragangliomas (PPGL) are rare neuroendocrine tumors characterized by a high frequency of hereditary forms. Based on transcriptome classification, PPGL can be classified in two different clusters. Cluster 1 tumors are caused by mutations in SDHx, VHL and FH genes and are characterized by a pseudohypoxic signature. Cluster 2 PPGL carry mutations in RET, NF1, MAX or TMEM127 genes and display an activation of the MAPK and mTOR signaling pathways. Many genetically engineered and allografted mouse models have been generated these past 30 years to investigate the mechanisms of PPGL tumorigenesis and test new therapeutic strategies. Among them, only Cluster 2-related models have been successful while no Cluster 1-related knockout mouse was so far reported to develop a PPGL. In this review, we present an overview of existing, successful or not, PPGL models, and a description of our own experience on the quest of Sdhb knockout mouse models of PPGL.


Subject(s)
Adrenal Gland Neoplasms/genetics , Neurofibromin 1/genetics , Pheochromocytoma/genetics , Succinate Dehydrogenase/genetics , Adrenal Gland Neoplasms/pathology , Animals , Humans , MAP Kinase Signaling System , Mice , Mice, Knockout , Mutation , Pheochromocytoma/pathology
10.
Nat Commun ; 6: 8784, 2015 Nov 02.
Article in English | MEDLINE | ID: mdl-26522426

ABSTRACT

The tricarboxylic acid (TCA) cycle is a central metabolic pathway responsible for supplying reducing potential for oxidative phosphorylation and anabolic substrates for cell growth, repair and proliferation. As such it thought to be essential for cell proliferation and tissue homeostasis. However, since the initial report of an inactivating mutation in the TCA cycle enzyme complex, succinate dehydrogenase (SDH) in paraganglioma (PGL), it has become clear that some cells and tissues are not only able to survive with a truncated TCA cycle, but that they are also able of supporting proliferative phenotype observed in tumours. Here, we show that loss of SDH activity leads to changes in the metabolism of non-essential amino acids. In particular, we demonstrate that pyruvate carboxylase is essential to re-supply the depleted pool of aspartate in SDH-deficient cells. Our results demonstrate that the loss of SDH reduces the metabolic plasticity of cells, suggesting vulnerabilities that can be targeted therapeutically.


Subject(s)
Electron Transport Complex II/metabolism , Membrane Proteins/metabolism , Neuroendocrine Tumors/enzymology , Paraganglioma/enzymology , Pyruvic Acid/metabolism , Succinate Dehydrogenase/metabolism , Animals , Aspartic Acid/metabolism , Electron Transport Complex II/genetics , Humans , Membrane Proteins/genetics , Mice , Mice, Knockout , Neuroendocrine Tumors/genetics , Neuroendocrine Tumors/metabolism , Oxidative Phosphorylation , Paraganglioma/genetics , Paraganglioma/metabolism , Succinate Dehydrogenase/genetics
11.
Oncotarget ; 6(32): 32955-65, 2015 Oct 20.
Article in English | MEDLINE | ID: mdl-26460615

ABSTRACT

Metastatic pheochromocytomas and paragangliomas (PPGL) are malignant neuroendocrine tumors frequently associated with germline mutations in the SDHB gene. SDHB-mutated PPGL display a hypermethylator phenotype associated with hallmarks of epithelial-to-mesenchymal transition (EMT). In the present study, we report the characterization of a unique model of Sdhb knockout in mouse chromaffin cells. Sdhb deficient cells exhibit a metastatic phenotype as highlighted by increased individual cell migration (characterized by faster motility and increased persistence) as well as high invasive and adhesion abilities. This phenotype is associated with the modulation of Twist1, Twist2, Tcf3, Snai1, N-cadherin or Krt19 expression, reflecting an EMT-like reprogramming of cells. Krt19 is epigenetically silenced in Sdhb-deficient cells and re-expressed after treatment by the demethylating agent decitabine. Krt19 rescue by lentiviral transduction in Sdhb-deficient cells and Krt19 inhibition by RNA interference in wild-type cells were performed. Both studies revealed the involvement of KRT19 in the invasive phenotype by modulating collective and individual migration and cell/extra-cellular matrix adhesion properties. These findings underline the role of hypermethylation and EMT in the in vitro acquisition of metastatic properties, following SDHB loss of function.


Subject(s)
Succinate Dehydrogenase/deficiency , Adrenal Gland Neoplasms/genetics , Adrenal Gland Neoplasms/metabolism , Animals , Cell Adhesion/physiology , Cell Line, Tumor , Chromaffin Cells/metabolism , Chromaffin Cells/pathology , Epithelial-Mesenchymal Transition , Humans , Mice , Mice, Knockout , Neoplasm Invasiveness , Neoplasm Metastasis , Paraganglioma/genetics , Paraganglioma/metabolism , Pheochromocytoma/genetics , Pheochromocytoma/metabolism , Succinate Dehydrogenase/genetics , Succinate Dehydrogenase/metabolism , Transcriptome
12.
Nat Commun ; 6: 6044, 2015 Jan 27.
Article in English | MEDLINE | ID: mdl-25625332

ABSTRACT

Pheochromocytomas and paragangliomas (PCCs/PGLs) are neural crest-derived tumours with a very strong genetic component. Here we report the first integrated genomic examination of a large collection of PCC/PGL. SNP array analysis reveals distinct copy-number patterns associated with genetic background. Whole-exome sequencing shows a low mutation rate of 0.3 mutations per megabase, with few recurrent somatic mutations in genes not previously associated with PCC/PGL. DNA methylation arrays and miRNA sequencing identify DNA methylation changes and miRNA expression clusters strongly associated with messenger RNA expression profiling. Overexpression of the miRNA cluster 182/96/183 is specific in SDHB-mutated tumours and induces malignant traits, whereas silencing of the imprinted DLK1-MEG3 miRNA cluster appears as a potential driver in a subgroup of sporadic tumours. Altogether, the complete genomic landscape of PCC/PGL is mainly driven by distinct germline and/or somatic mutations in susceptibility genes and reveals different molecular entities, characterized by a set of unique genomic alterations.


Subject(s)
Adrenal Gland Neoplasms/genetics , Genetic Predisposition to Disease , Genome, Human/genetics , Genomics/methods , Mutation/genetics , Paraganglioma/genetics , Pheochromocytoma/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Child , Cohort Studies , DNA Copy Number Variations , DNA Methylation/genetics , Exome/genetics , Female , Gene Expression Profiling , Humans , Male , MicroRNAs/genetics , MicroRNAs/metabolism , Middle Aged , Sequence Analysis, DNA , Young Adult
13.
Int J Cancer ; 135(10): 2237-48, 2014 Nov 15.
Article in English | MEDLINE | ID: mdl-25124653

ABSTRACT

Although the alteration of cellular metabolism in cancer was reported by Warburg in the early 1930s, a regain of interest in cancer metabolism has more recently followed the discovery of germline or somatic mutations in genes coding for metabolic enzymes (succinate dehydrogenase, fumarate hydratase and isocitrate dehydrogenase) that are associated with tumor susceptibility. Mutations in these genes are found in numerous tumor types including paragangliomas, kidney cancers, leiomyomas, glioblastomas and acute myeloid leukemia. They lead to the accumulation of so-called oncometabolites that behave as competitors of 2-oxoglutarate-dependent dioxygenases, involved in a broad spectrum of pathways such as hypoxic response and epigenetic reprogramming. Here, we review the diverse pathways affected by oncometabolites, their potential role in cancer formation, maintenance, metastasis and sensitivity to chemotherapies, as well as emerging new therapeutic strategies.


Subject(s)
Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Metabolome , Neoplasms/genetics , Neoplasms/therapy , Humans , Neoplasms/metabolism
14.
Hum Mol Genet ; 23(9): 2440-6, 2014 May 01.
Article in English | MEDLINE | ID: mdl-24334767

ABSTRACT

Malignant pheochromocytoma (PCC) and paraganglioma (PGL) are mostly caused by germline mutations of SDHB, encoding a subunit of succinate dehydrogenase. Using whole-exome sequencing, we recently identified a mutation in the FH gene encoding fumarate hydratase, in a PCC with an 'SDH-like' molecular phenotype. Here, we investigated the role of FH in PCC/PGL predisposition, by screening for germline FH mutations in a large international cohort of patients. We screened 598 patients with PCC/PGL without mutations in known PCC/PGL susceptibility genes. We searched for FH germline mutations and large deletions, by direct sequencing and multiplex ligation-dependent probe amplification methods. Global alterations in DNA methylation and protein succination were assessed by immunohistochemical staining for 5-hydroxymethylcytosine (5-hmC) and S-(2-succinyl) cysteine (2SC), respectively. We identified five pathogenic germline FH mutations (four missense and one splice mutation) in five patients. Somatic inactivation of the second allele, resulting in a loss of fumarate hydratase activity, was demonstrated in tumors with FH mutations. Low tumor levels of 5-hmC, resembling those in SDHB-deficient tumors, and positive 2SC staining were detected in tumors with FH mutations. Clinically, metastatic phenotype (P = 0.007) and multiple tumors (P = 0.02) were significantly more frequent in patients with FH mutations than those without such mutations. This study reveals a new role for FH in susceptibility to malignant and/or multiple PCC/PGL. Remarkably, FH-deficient PCC/PGLs display the same pattern of epigenetic deregulation as SDHB-mutated malignant PCC/PGL. Therefore, we propose that mutation screening for FH should be included in PCC/PGL genetic testing, at least for tumors with malignant behavior.


Subject(s)
Fumarate Hydratase/genetics , Germ-Line Mutation/genetics , Paraganglioma/genetics , Pheochromocytoma/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Child , Exons/genetics , Female , Genetic Predisposition to Disease/genetics , Humans , Male , Middle Aged , Young Adult
15.
Transcription ; 4(2): 67-71, 2013.
Article in English | MEDLINE | ID: mdl-23412358

ABSTRACT

The clinical importance of tumor suppressor p53 makes it one of the most studied transcription factors. A comparison of mammalian p53 transcriptional repertoires may help identify fundamental principles in genome evolution and better understand cancer processes. Here we summarize mechanisms underlying the divergence of mammalian p53 transcriptional repertoires, with an emphasis on the rapid evolution of fuzzy tandem repeats containing p53 response elements.


Subject(s)
Tumor Suppressor Protein p53/metabolism , Animals , Evolution, Molecular , Humans , Mice , Nucleotide Motifs , Promoter Regions, Genetic , Response Elements , Retinoblastoma-Like Protein p130/metabolism , Tandem Repeat Sequences , Tumor Suppressor Protein p53/genetics
16.
PLoS One ; 7(8): e42252, 2012.
Article in English | MEDLINE | ID: mdl-22879924

ABSTRACT

Almost all prostate cancers respond to androgen deprivation treatment but many recur. We postulated that risk of hormone escape--frequency and delay--are influenced by hormone therapy modalities. More, hormone therapies induce crucial biological changes involving androgen receptors; some might be targets for escape prevention. We investigated the relationship between the androgen deprivation treatment and the risk of recurrence using nude mice bearing the high grade, hormone-dependent human prostate cancer xenograft PAC120. Tumor-bearing mice were treated by Luteinizing-Hormone Releasing Hormone (LHRH) antagonist alone, continuous or intermittent regimen, or combined with androgen receptor (AR) antagonists (bicalutamide or flutamide). Tumor growth was monitored. Biological changes were studied as for genomic alterations, AR mutations and protein expression in a large series of recurrent tumors according to hormone therapy modalities. Therapies targeting Her-2 or AKT were tested in combination with castration. All statistical tests were two-sided. Tumor growth was inhibited by continuous administration of the LH-RH antagonist degarelix (castration), but 40% of tumors recurred. Intermittent castration or complete blockade induced by degarelix and antiandrogens combination, inhibited tumor growth but increased the risk of recurrence (RR) as compared to continuous castration (RR(intermittent): 14.5, RR(complete blockade): 6.5 and 1.35). All recurrent tumors displayed new quantitative genetic alterations and AR mutations, whatever the treatment modalities. AR amplification was found after complete blockade. Increased expression of Her-2/neu with frequent ERK/AKT activation was detected in all variants. Combination of castration with a Her-2/neu inhibitor decreased recurrence risk (0.17) and combination with an mTOR inhibitor prevented it. Anti-hormone treatments influence risk of recurrence although tumor growth inhibition was initially similar. Recurrent tumors displayed genetic instability, AR mutations, and alterations of phosphorylation pathways. We postulated that Her-2/AKT pathways allowed salvage of tumor cells under castration and we demonstrated that their inhibition prevented tumor recurrence in our model.


Subject(s)
Androgens/therapeutic use , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/enzymology , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Protein-Tyrosine Kinases/antagonists & inhibitors , Xenograft Model Antitumor Assays , Androgens/deficiency , Animals , Base Sequence , Castration , Cluster Analysis , Combined Modality Therapy , Disease-Free Survival , Gene Dosage/genetics , Humans , Male , Mice , Mutation/genetics , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Protein-Tyrosine Kinases/metabolism , Receptor, ErbB-2/genetics , Receptors, Androgen/genetics
17.
Chemistry ; 18(40): 12834-44, 2012 Oct 01.
Article in English | MEDLINE | ID: mdl-22907863

ABSTRACT

This work reveals the preponderance of an intramolecular metal chelation phenomenon in a controlled radical polymerization system involving the reversible trapping of the radical chains by a cobalt complex bis(acetylacetonato)cobalt(II). The cobalt-mediated radical polymerization (CMRP) of a series of N-vinyl amides was considered with the aim of studying the effect of the cobalt chelation by the amide moiety of the last monomer unit of the chain. The latter reinforces the cobalt-polymer bond in the order N-vinylpyrrolidone

18.
PLoS Genet ; 8(6): e1002731, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22761580

ABSTRACT

Evolutionary forces that shape regulatory networks remain poorly understood. In mammals, the Rb pathway is a classic example of species-specific gene regulation, as a germline mutation in one Rb allele promotes retinoblastoma in humans, but not in mice. Here we show that p53 transactivates the Retinoblastoma-like 2 (Rbl2) gene to produce p130 in murine, but not human, cells. We found intronic fuzzy tandem repeats containing perfect p53 response elements to be important for this regulation. We next identified two other murine genes regulated by p53 via fuzzy tandem repeats: Ncoa1 and Klhl26. The repeats are poorly conserved in evolution, and the p53-dependent regulation of the murine genes is lost in humans. Our results indicate a role for the rapid evolution of tandem repeats in shaping differences in p53 regulatory networks between mammalian species.


Subject(s)
Gene Expression Regulation , Retinoblastoma-Like Protein p130 , Retinoblastoma/genetics , Tandem Repeat Sequences/genetics , Tumor Suppressor Protein p53 , Animals , Cells, Cultured , Evolution, Molecular , Fibroblasts/cytology , Fibroblasts/metabolism , Humans , Introns/genetics , Mice , Mutation , Nuclear Receptor Coactivator 1/genetics , Nuclear Receptor Coactivator 1/metabolism , Response Elements/genetics , Retinoblastoma-Like Protein p130/genetics , Retinoblastoma-Like Protein p130/metabolism , Species Specificity , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism
19.
FASEB J ; 26(1): 460-7, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21982950

ABSTRACT

Markers of prostate tumor recurrence after radical prostatectomy are lacking and highly demanded. The androgen receptor (AR) is a nuclear receptor that plays a pivotal role in normal and cancerous prostate tissue. AR interacts with a number of proteins modulating its stability, localization, and activity. To test the hypothesis that an increased expression of AR partners might foster tumor development, we immunopurified AR partners in human tumors xenografted into mice. One of the identified AR partners was the multifunctional enzyme carbamoyl-phosphate synthetase II, aspartate transcarbamylase, and dihydroorotase (CAD), which catalyzes the 3 initial steps of pyrimidine biosynthesis. We combined experiments in C4-2, LNCaP, 22RV1, and PC3 human prostate cell lines and analysis of frozen radical prostatectomy samples to study the CAD-AR interaction. We show here that in prostate tumor cells, CAD fosters AR translocation into the nucleus and stimulates its transcriptional activity. Notably, in radical prostatectomy specimens, CAD expression was not correlated with proliferation markers, but a higher CAD mRNA level was associated with local tumor extension (P=0.049) and cancer relapse (P=0.017). These results demonstrate an unsuspected function for a key metabolic enzyme and identify CAD as a potential predictive marker of cancer relapse.


Subject(s)
Aspartate Carbamoyltransferase/metabolism , Biomarkers, Tumor/metabolism , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/metabolism , Dihydroorotase/metabolism , Neoplasm Recurrence, Local/diagnosis , Prostatic Neoplasms/diagnosis , Receptors, Androgen/metabolism , Androgens/metabolism , Animals , Aspartate Carbamoyltransferase/genetics , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/genetics , Cell Line, Tumor , Cell Nucleus/metabolism , Cytosol/metabolism , Dihydroorotase/genetics , Humans , Male , Mice , Neoplasm Recurrence, Local/metabolism , Neoplasm Transplantation , Predictive Value of Tests , Prostatic Neoplasms/metabolism , Pyrimidines/biosynthesis , RNA, Small Interfering/pharmacology , Receptors, Androgen/genetics , Transcription, Genetic/physiology , Transplantation, Heterologous
20.
Cancer Res ; 71(24): 7649-58, 2011 Dec 15.
Article in English | MEDLINE | ID: mdl-22037878

ABSTRACT

The antitumor effects of pharmacologic inhibitors of angiogenesis are hampered in patients by the rapid development of tumor resistance, notably through increased invasiveness and accelerated metastasis. Here, we reevaluated the role of the endogenous antiangiogenic thrombospondin 1 (TSP1) in prostate carcinomas in which angiogenesis is an active process. In xenografted tumors, we observed that TSP1 altogether inhibited angiogenesis and fostered tumor development. Our results show that TSP1 is a potent stimulator of prostate tumor cell migration. This effect required CD36, which also mediates TSP1 antiangiogenic activity, and was mimicked by an antiangiogenic TSP1-derived peptide. As suspected for pharmacologic inhibitors of angiogenesis, the TSP1 capacities to increase hypoxia and to trigger cell migration are thus inherently linked. Importantly, although antiangiogenic TSP1 increases hypoxia in vivo, our data show that, in turn, hypoxia induced TSP1, thus generating a vicious circle in prostate tumors. In radical prostatectomy specimens, we found TSP1 expression significantly associated with invasive tumors and with tumors which eventually recurred. TSP1 may thus help select patients at risk of prostate-specific antigen relapse. Together, the data suggest that intratumor disruption of the hypoxic cycle through TSP1 silencing will limit tumor invasion.


Subject(s)
Cell Movement , Neovascularization, Pathologic/genetics , Prostatic Neoplasms/genetics , RNA Interference , Thrombospondin 1/genetics , Animals , CD36 Antigens/metabolism , Calcium/metabolism , Cell Hypoxia , Cell Line, Tumor , Enzyme-Linked Immunosorbent Assay , Gene Expression Regulation, Neoplastic , Humans , Hypoxia , Male , Mice , Mice, Nude , Neoplasm Invasiveness , Neoplasm Recurrence, Local , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Orchiectomy , Peptides/pharmacology , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Reverse Transcriptase Polymerase Chain Reaction , TRPV Cation Channels/metabolism , Thrombospondin 1/chemistry , Thrombospondin 1/metabolism , Tumor Burden/drug effects , Xenograft Model Antitumor Assays/methods
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