Etablissement d'un nouveau modèle pérclinique de cancer de la prostate et identification de biomarqueurs de résistance au docetaxel

One of the major hindrances in the study of the biology of prostate cancer is the limited number of laboratory models. Most of these models have been obtained from prostate tumor metastases or have been artificially established in vitro.We recently developed one new cell line (IGR-CaP1) derived from patients with clinically localized prostate cancer. In contrast to previously established models from metastases tissues, IGR-CaP1 may be a suitable model to study molecular pathways implicated in the early steps of the oncogenic development of prostate cancer. Furthermore, its high tumorigenic properties and its ability to induce mixed bone lesions, make it as a potential model for both tumor progression and drug assessment in animals.Docetaxel is the standard treatment for metastatic castration-resistant prostate cancer (CRPC) since 2004. In spite of a benefit in survival, drug resistance is often observed. Therefore, it is crucial to identify predictive markers to select patients who will respond to docetaxel. In order to investigate mechanisms of docetaxel resistance, we derived docetaxel-resistant variants from the IGR-CaP1 human prostate cancer cell line. A microarray genomic analysis comparing chemo-resistant versus sensitive prostate cell lines was used to identify a signature of genes potentially implicated in docetaxel resistance. Among these genes, we focused on LZTS1 wich is underexpressed in IGR-CaP1 resistant variants. LZTS1 is a tumor suppressor that controls the cell cycle by interacting with Cdc25C. Our data suggest that depletion of LZTS1 is potentially involved in the mecanism of docetaxel resistance. Finally, an immunohistochemical analysis will be done on human biopsies from the phase III GETUG12 trial patients. Ultimately, our study could help to improve selection of patients that could benefit from docetaxel chemotherapy.La mise au point de modèles de laboratoire est d’une importance cruciale pour comprendre la biologie du cancer de la prostate, ainsi que pour évaluer les nouveaux traitements. Le développement de tels modèles est particulièrement difficile et reste à ce jour insuffisant car la majorité de ces modèles est d’origine métastatique ou obtenu in vitro d’une façon artificielle. C’est pourquoi, nous avons entrepris au laboratoire, l’établissement de nouveaux modèles à partir d’un cancer primaire de prostate tumorale et obtenu la lignée IGR-CaP1. La lignée IGR-CaP1 constitue un modèle adapté pour étudier les étapes précoces de la cancérogenèse prostatique. De plus, sa tumoroginicité et sa capacité à induire des métastases osseuses de nature mixtes ostéoblastiques et ostéolytiques font de ce modèles un outil potentiellement intéressant pour étudier les mécanismes métastatiques et rechercher de nouvelles cibles thérapeutiques. Depuis 2004, le traitement de référence des cancers de la prostate métastatiques hormono-résistants est une chimiothérapie par le Docetaxel. Cependant, malgré le bénéfice de survie obtenu, presque la moitié des patients traités par le Docetaxel développent une résistance à la chimiothérapie. Il est donc urgent d’identifier un biomarqueur prédictif pour sélectionner les patients qui vont bénéficier de cette chimiothérapie afin de contourner cette résistance. Dans le but d’étudier les mécanismes de résistance au Docetaxel dans le cancer de la prostate, nous avons établi plusieurs clones résistants au Docetaxel à partir de la lignée IGR-CaP1. Ces clones résistants nous ont permis de réaliser une analyse génomique à haut-débit par microarray comparant l’expression génique entre la lignée sensible et les clones résistants et d’identifier une signature de gènes potentiellement impliqués dans la résistance au Docetaxel. Parmi les gènes identifiés, nous nous sommes focalisés sur le gène LZTS1 sous-exprimé dans tous les clones résistants. LZTS1 est un suppresseur de tumeur qui contrôle le cycle cellulaire en interagissant avec la cycline Cdc25C. Nos résultats suggèrent que la déplétion de LZTS1 est potentiellement impliquée dans le mécanisme de résistance au Docetaxel. La finalité de notre projet est de valider nos résultats par immunohistochimie à partir des prélèvements tumoraux obtenus dans l’essai de phase III GETUG12. Nous espérons que notre étude permettra aux cliniciens de sélectionner les sous-groupes de patients susceptibles de profiter d’un traitement par Docetaxel

Etablissement d'un nouveau modèle pérclinique de cancer de la prostate et identification de biomarqueurs de résistance au docetaxel

One of the major hindrances in the study of the biology of prostate cancer is the limited number of laboratory models. Most of these models have been obtained from prostate tumor metastases or have been artificially established in vitro.We recently developed one new cell line (IGR-CaP1) derived from patients with clinically localized prostate cancer. In contrast to previously established models from metastases tissues, IGR-CaP1 may be a suitable model to study molecular pathways implicated in the early steps of the oncogenic development of prostate cancer. Furthermore, its high tumorigenic properties and its ability to induce mixed bone lesions, make it as a potential model for both tumor progression and drug assessment in animals.Docetaxel is the standard treatment for metastatic castration-resistant prostate cancer (CRPC) since 2004. In spite of a benefit in survival, drug resistance is often observed. Therefore, it is crucial to identify predictive markers to select patients who will respond to docetaxel. In order to investigate mechanisms of docetaxel resistance, we derived docetaxel-resistant variants from the IGR-CaP1 human prostate cancer cell line. A microarray genomic analysis comparing chemo-resistant versus sensitive prostate cell lines was used to identify a signature of genes potentially implicated in docetaxel resistance. Among these genes, we focused on LZTS1 wich is underexpressed in IGR-CaP1 resistant variants. LZTS1 is a tumor suppressor that controls the cell cycle by interacting with Cdc25C. Our data suggest that depletion of LZTS1 is potentially involved in the mecanism of docetaxel resistance. Finally, an immunohistochemical analysis will be done on human biopsies from the phase III GETUG12 trial patients. Ultimately, our study could help to improve selection of patients that could benefit from docetaxel chemotherapy.La mise au point de modèles de laboratoire est d’une importance cruciale pour comprendre la biologie du cancer de la prostate, ainsi que pour évaluer les nouveaux traitements. Le développement de tels modèles est particulièrement difficile et reste à ce jour insuffisant car la majorité de ces modèles est d’origine métastatique ou obtenu in vitro d’une façon artificielle. C’est pourquoi, nous avons entrepris au laboratoire, l’établissement de nouveaux modèles à partir d’un cancer primaire de prostate tumorale et obtenu la lignée IGR-CaP1. La lignée IGR-CaP1 constitue un modèle adapté pour étudier les étapes précoces de la cancérogenèse prostatique. De plus, sa tumoroginicité et sa capacité à induire des métastases osseuses de nature mixtes ostéoblastiques et ostéolytiques font de ce modèles un outil potentiellement intéressant pour étudier les mécanismes métastatiques et rechercher de nouvelles cibles thérapeutiques. Depuis 2004, le traitement de référence des cancers de la prostate métastatiques hormono-résistants est une chimiothérapie par le Docetaxel. Cependant, malgré le bénéfice de survie obtenu, presque la moitié des patients traités par le Docetaxel développent une résistance à la chimiothérapie. Il est donc urgent d’identifier un biomarqueur prédictif pour sélectionner les patients qui vont bénéficier de cette chimiothérapie afin de contourner cette résistance. Dans le but d’étudier les mécanismes de résistance au Docetaxel dans le cancer de la prostate, nous avons établi plusieurs clones résistants au Docetaxel à partir de la lignée IGR-CaP1. Ces clones résistants nous ont permis de réaliser une analyse génomique à haut-débit par microarray comparant l’expression génique entre la lignée sensible et les clones résistants et d’identifier une signature de gènes potentiellement impliqués dans la résistance au Docetaxel. Parmi les gènes identifiés, nous nous sommes focalisés sur le gène LZTS1 sous-exprimé dans tous les clones résistants. LZTS1 est un suppresseur de tumeur qui contrôle le cycle cellulaire en interagissant avec la cycline Cdc25C. Nos résultats suggèrent que la déplétion de LZTS1 est potentiellement impliquée dans le mécanisme de résistance au Docetaxel. La finalité de notre projet est de valider nos résultats par immunohistochimie à partir des prélèvements tumoraux obtenus dans l’essai de phase III GETUG12. Nous espérons que notre étude permettra aux cliniciens de sélectionner les sous-groupes de patients susceptibles de profiter d’un traitement par Docetaxel

Establishment of a new preclinical human prostate cancer model and identification of docetaxel-resistance biomarker

La mise au point de modèles de laboratoire est d’une importance cruciale pour comprendre la biologie du cancer de la prostate, ainsi que pour évaluer les nouveaux traitements. Le développement de tels modèles est particulièrement difficile et reste à ce jour insuffisant car la majorité de ces modèles est d’origine métastatique ou obtenu in vitro d’une façon artificielle. C’est pourquoi, nous avons entrepris au laboratoire, l’établissement de nouveaux modèles à partir d’un cancer primaire de prostate tumorale et obtenu la lignée IGR-CaP1. La lignée IGR-CaP1 constitue un modèle adapté pour étudier les étapes précoces de la cancérogenèse prostatique. De plus, sa tumoroginicité et sa capacité à induire des métastases osseuses de nature mixtes ostéoblastiques et ostéolytiques font de ce modèles un outil potentiellement intéressant pour étudier les mécanismes métastatiques et rechercher de nouvelles cibles thérapeutiques. Depuis 2004, le traitement de référence des cancers de la prostate métastatiques hormono-résistants est une chimiothérapie par le Docetaxel. Cependant, malgré le bénéfice de survie obtenu, presque la moitié des patients traités par le Docetaxel développent une résistance à la chimiothérapie. Il est donc urgent d’identifier un biomarqueur prédictif pour sélectionner les patients qui vont bénéficier de cette chimiothérapie afin de contourner cette résistance. Dans le but d’étudier les mécanismes de résistance au Docetaxel dans le cancer de la prostate, nous avons établi plusieurs clones résistants au Docetaxel à partir de la lignée IGR-CaP1. Ces clones résistants nous ont permis de réaliser une analyse génomique à haut-débit par microarray comparant l’expression génique entre la lignée sensible et les clones résistants et d’identifier une signature de gènes potentiellement impliqués dans la résistance au Docetaxel. Parmi les gènes identifiés, nous nous sommes focalisés sur le gène LZTS1 sous-exprimé dans tous les clones résistants. LZTS1 est un suppresseur de tumeur qui contrôle le cycle cellulaire en interagissant avec la cycline Cdc25C. Nos résultats suggèrent que la déplétion de LZTS1 est potentiellement impliquée dans le mécanisme de résistance au Docetaxel. La finalité de notre projet est de valider nos résultats par immunohistochimie à partir des prélèvements tumoraux obtenus dans l’essai de phase III GETUG12. Nous espérons que notre étude permettra aux cliniciens de sélectionner les sous-groupes de patients susceptibles de profiter d’un traitement par Docetaxel.One of the major hindrances in the study of the biology of prostate cancer is the limited number of laboratory models. Most of these models have been obtained from prostate tumor metastases or have been artificially established in vitro.We recently developed one new cell line (IGR-CaP1) derived from patients with clinically localized prostate cancer. In contrast to previously established models from metastases tissues, IGR-CaP1 may be a suitable model to study molecular pathways implicated in the early steps of the oncogenic development of prostate cancer. Furthermore, its high tumorigenic properties and its ability to induce mixed bone lesions, make it as a potential model for both tumor progression and drug assessment in animals.Docetaxel is the standard treatment for metastatic castration-resistant prostate cancer (CRPC) since 2004. In spite of a benefit in survival, drug resistance is often observed. Therefore, it is crucial to identify predictive markers to select patients who will respond to docetaxel. In order to investigate mechanisms of docetaxel resistance, we derived docetaxel-resistant variants from the IGR-CaP1 human prostate cancer cell line. A microarray genomic analysis comparing chemo-resistant versus sensitive prostate cell lines was used to identify a signature of genes potentially implicated in docetaxel resistance. Among these genes, we focused on LZTS1 wich is underexpressed in IGR-CaP1 resistant variants. LZTS1 is a tumor suppressor that controls the cell cycle by interacting with Cdc25C. Our data suggest that depletion of LZTS1 is potentially involved in the mecanism of docetaxel resistance. Finally, an immunohistochemical analysis will be done on human biopsies from the phase III GETUG12 trial patients. Ultimately, our study could help to improve selection of patients that could benefit from docetaxel chemotherapy

Oncofetal Chondroitin Sulfate: A Putative Therapeutic Target in Adult and Pediatric Solid Tumors

Solid tumors remain a major challenge for targeted therapeutic intervention strategies such as antibody-drug conjugates and immunotherapy. At a minimum, clear and actionable solid tumor targets have to comply with the key biological requirement of being differentially over-expressed in solid tumors and metastasis, in contrast to healthy organs. Oncofetal chondroitin sulfate is a cancer-specific secondary glycosaminoglycan modification to proteoglycans expressed in a variety of solid tumors and metastasis. Normally, this modification is found to be exclusively expressed in the placenta, where it is thought to facilitate normal placental implantation during pregnancy. Informed by this biology, oncofetal chondroitin sulfate is currently under investigation as a broad and specific target in solid tumors. Here, we discuss oncofetal chondroitin sulfate as a potential therapeutic target in childhood solid tumors in the context of current knowhow obtained over the past five years in adult cancers.Medicine, Faculty ofOther UBCNon UBCPathology and Laboratory Medicine, Department ofUrologic Sciences, Department ofReviewedFacult

Hepatoma-derived growth factor-related protein 2 promotes DNA repair by homologous recombination

We have recently identified lens epithelium-derived growth factor (LEDGF/p75, also known as PSIP1) as a component of the homologous recombination DNA repair machinery. Through its Pro-Trp-Trp-Pro (PWWP) domain, LEDGF/p75 binds to histone marks associated with active transcription and promotes DNA end resection by recruiting DNA endonuclease retinoblastoma-binding protein 8 (RBBP8/CtIP) to broken DNA ends. Here we show that the structurally related PWWP domain-containing protein, hepatoma-derived growth factor-related protein 2 (HDGFRP2), serves a similar function in homologous recombination repair. Its depletion compromises the survival of human U2OS osteosarcoma and HeLa cervix carcinoma cells and impairs the DNA damage-induced phosphorylation of replication protein A2 (RPA2) and the recruitment of DNA endonuclease RBBP8/CtIP to DNA double strand breaks. In contrast to LEDGF/p75, HDGFRP2 binds preferentially to histone marks characteristic for transcriptionally silent chromatin. Accordingly, HDGFRP2 is found in complex with the heterochromatin-binding chromobox homologue 1 (CBX1) and Pogo transposable element with ZNF domain (POGZ). Supporting the functionality of this complex, POGZ-depleted cells show a similar defect in DNA damage-induced RPA2 phosphorylation as HDGFRP2-depleted cells. These data suggest that HDGFRP2, possibly in complex with POGZ, recruits homologous recombination repair machinery to damaged silent genes or to active genes silenced upon DNA damage

Targeting CDC25C, PLK1 and CHEK1 to overcome Docetaxel resistance induced by loss of LZTS1 in prostate cancer

International audienceDocetaxel is used as a standard treatment in patients with metastatic castration-resistant prostate cancer. However, a large subset of patients develops resistance. Understanding resistance mechanisms, which are largely unknown, will allow identification of predictive biomarkers and therapeutic targets. We established resistant IGR-CaP1 prostate cancer cell lines for different doses of Docetaxel. We investigated gene expression profiles by microarray analyses in these cell lines and generated a signature of 99 highly differentially expressed genes potentially implicated in chemoresistance. We focused on the role of the cell cycle regulator LZTS1, which was under-expressed in the Docetaxel-resistant cell lines, its inhibition resulting from the promoter methylation. Knockdown of LZTS1 in parental cells with siRNA showed that LZTS1 plays a role in the acquisition of the resistant phenotype. Furthermore, we observed that targeting CDC25C, a partner of LZTS1, with the NSC663284 inhibitor specifically killed the Docetaxel-resistant cells. To further investigate the role of CDC25C, we used inhibitors of the mitotic kinases that regulate CDC25C. Inhibition of CHEK1 and PLK1 induced growth arrest and cell death in the resistant cells. Our findings identify an important role of LZTS1 through its regulation of CDC25C in Docetaxel resistance in prostate cancer and suggest that CDC25C, or the mitotic kinases CHEK1 and PLK1, could be efficient therapeutic targets to overcome Docetaxel resistance

The IGR-CaP1 Xenograft Model Recapitulates Mixed Osteolytic/Blastic Bone Lesions Observed in Metastatic Prostate Cancer12

Bone metastases have a devastating impact on quality of life and bone pain in patients with prostate cancer and decrease survival. Animal models are important tools in investigating the pathogenesis of the disease and in developing treatment strategies for bone metastases, but few animal models recapitulate spontaneous clinical bone metastatic spread. In the present study, IGR-CaP1, a new cell line derived from primary prostate cancer, was stably transduced with a luciferase-expressing viral vector to monitor tumor growth in mice using bioluminescence imaging. The IGR-CaP1 tumors grew when subcutaneously injected or when orthotopically implanted, reconstituted the prostate adenocarcinoma with glandular acini-like structures, and could disseminate to the liver and lung. Bone lesions were detected using bioluminescence imaging after direct intratibial or intracardiac injections. Anatomic bone structure assessed using high-resolution computed tomographic scans showed both lytic and osteoblastic lesions. Technetium Tc 99m methylene diphosphonate micro single-photon emission computed tomography confirmed the mixed nature of the lesions and the intensive bone remodeling. We also identified an expression signature for responsiveness of IGR-CaP1 cells to the bone microenvironment, namely expression of CXCR4, MMP-9, Runx2, osteopontin, osteoprotegerin, ADAMTS14, FGFBP2, and HBB. The IGR-CaP1 cell line is a unique model derived from a primary tumor, which can reconstitute human prostate adenocarcinoma in animals and generate experimental bone metastases, providing a novel means for understanding the mechanisms of bone metastasis progression and allowing preclinical testing of new therapies

Stemness markers characterize IGR-CaP1, a new cell line derived from primary epithelial prostate cancer

International audienceDeciphering molecular pathways involved in the early steps of prostate oncogenesis requires both in vitro and in vivo models derived from human primary tumors. However the few recognized models of human prostate epithelial cancer originate from metastases. To date, very few models are proposed from primary tumors and immortalizing normal human prostate cells does not recapitulate the natural history of the disease. By culturing human prostate primary tumor cells onto human epithelial extra-cellular matrix, we successfully selected a new prostate cancer cell line, IGR-CaP1, and clonally-derived subclones. IGR-CaP1 cells, that harbor a tetraploid karyotype, high telomerase activity and mutated TP53, rapidly induced subcutaneous xenografts in nude mice. Furthermore, IGR-CaP1 cell lines, all exhibiting negativity for the androgen receptor and PSA, express the specific prostate markers alpha-methylacyl-CoA racemase and a low level of the prostate-specific membrane antigen PSMA, along with the prostate basal epithelial markers CK5 and CK14. More importantly, these clones express high CD44, CD133, and CXCR4 levels associated with high expression of α2β1-integrin and Oct4 which are reported to be prostate cancer stemness markers. RT-PCR data also revealed high activation of the Sonic Hedgehog signalling pathway in these cells. Additionally, the IGR-CaP1 cells possess a 3D sphere-forming ability and a renewal capacity by maintaining their CSC potential after xenografting in mice. As a result, the hormone-independent IGR-CaP1 cellular clones exhibit the original features of both basal prostate tissue and cancer stemness. Tumorigenic IGR-CaP1 clones constitute invaluable human models for studying prostate cancer progression and drug assessment in vitro as well as in animals specifically for developing new therapeutic approaches targeting prostate cancer stem cells

Clusterin knockdown sensitizes prostate cancer cells to taxane by modulating mitosis

Clusterin (CLU) is a stress‐activated molecular chaperone that confers treatment resistance to taxanes when highly expressed. While CLU inhibition potentiates activity of taxanes and other anti‐cancer therapies in preclinical models, progression to treatment‐resistant disease still occurs implicating additional compensatory survival mechanisms. Taxanes are believed to selectively target cells in mitosis, a complex mechanism controlled in part by balancing antagonistic roles of Cdc25C and Wee1 in mitosis progression. Our data indicate that CLU silencing induces a constitutive activation of Cdc25C, which delays mitotic exit and hence sensitizes cancer cells to mitotic‐targeting agents such as taxanes. Unchecked Cdc25C activation leads to mitotic catastrophe and cell death unless cells up‐regulate protective mechanisms mediated through the cell cycle regulators Wee1 and Cdk1. In this study, we show that CLU silencing induces a constitutive activation of Cdc25C via the phosphatase PP2A leading to relief of negative feedback inhibition and activation of Wee1‐Cdk1 to promote survival and limit therapeutic efficacy. Simultaneous inhibition of CLU‐regulated cell cycle effector Wee1 may improve synergistic responses of biologically rational combinatorial regimens using taxanes and CLU inhibitors