The “Aging, Prenylation and Cancer” team, gathers 4 groups with distinct but tightly linked research axes oriented on aging, cancer, spermatogenesis and networks/systems biology.
The involvement of aberrantly accumulated or activated prenylated proteins (with Farnesyl or Geranyl‐geranyl hydrophobic C terminus anchors) in all these disease categories (genetically determined premature aging (progeroid) syndromes (PS) and lipodystrophies, cancer, human physiological and pathological spermatogenesis) serves as a focus and common denominator of our scientific research strategies. The department “Aging, Prenylation & Cancer” is unique in France and will increase collaborations with French, EU and international teams currently working on premature aging, laminopathies and prenylated proteins-associated cancers with the major aim of developing translational research.
We propose to use the exploration of both in vitro and in vivo models:
1) to further delineate and enlarge the clinical spectrum of diseases characterized by premature aging/ lipodystrophy/cancer development/altered human spermiogenesis, possibly associated to dysfunction of prenylated proteins or functional interactors;
2) to dissect the molecular mechanisms underlying these syndromes, allowing to identify therapeutic targets;
3) to develop pre-clinical proofs of concepts;
4) to extrapolate the generated knowledge aiming to improve healthy aging, by verifying whether the identified mechanisms apply to similar “natural” aging-associated morbidities;
5) to improve our knowledge on cancers involving prenylated proteins’ dysfunction towards identifying therapeutic targets and ameliorate patients’ healthcare;
6) to launch novel therapeutic trials.
The main long-term objective of the team is indeed the transposition of our discoveries to address and implement care in premature aging syndromes, age-related morbidities, natural aging and cancers. A strategy combining several approaches will lead to the pursuit of the following objectives in the field of “Prenylopathies”.
Since we and others discovered the involvement of the LMNA gene encoding the nuclear proteins Lamins A/C, in Hutchinson-Gilford Progeria Syndrome (OMIM #176670), one of the best known premature aging syndromes (De Sandre-Giovannoli et al., Science 2003), our team focuses on translational research on premature aging (progeroid) syndromes (PS), nuclear protein-linked lipodystrophies, as well as other premature aging diseases, with or without an identified molecular basis. Our research aims to identify novel genes involved in these syndromes, increase our understanding of their pathophysiological mechanisms and to develop new therapeutic approaches using patients’ cell lines or mouse models as our main preclinical tools.
PS are a group of rare genetic disorders characterized by many clinical features that mimic those associated to physiological aging. Many PS share the dysfunction of a prenylated protein. This is the case in Hutchinson-Gilford Progeria Syndrome (HGPS) and related PS, where prenylated progerin or prelamin A aberrantly accumulate in cells’ nuclei, exerting multiple toxic effects and ultimately leading to the systemic severe phenotypes observed in patients. Besides, several other PS have been discovered as being caused by activating mutations in genes encoding permanently prenylated proteins of the RAS‐MAPK pathways. Among these “RASopathies”, research on Costello syndrome, due to H-RAS mutations, will be part of this project. Finally, several lines of evidence suggest that progerin production may also be involved in natural aging pathomechanisms.
a. Extend the clinical spectrum of syndromes associated with prelamin A deficient processing.
We identified several novel or related syndromes mainly characterized by premature aging 7-25 or lipodystrophy / metabolic syndrome 9, 26 linked to mutations of LMNA or functionally related genes; as well as previously undiscovered genetic associations 10.
b. Produce in vitro and in vivo models for studying rare progeroid syndromes
A large panel of data and primary fibroblast cell lines from children affected with typical Progeria or atypical PS, and progeroid RASopathies have been collected through national and international collaborations and are stored in the Biological Resources Center (CRB-TAC), at La Timone hospital, Marseille. Human iPS cell lines (hiPSC) have also been derived and used to study the pathophysiology of the syndrome 27 or compare ongoing or proposed treatments 28. Primary endothelial cells and progenitors issued from human umbilical vein or cord blood were also used as in vitro models to study HGPS pathophysiology 29. Importantly, in collaboration with Pr Lopez-Otin in Oviedo, we generated the mouse model of progeria which most closely mimics the human disease (Knock-In LmnaG609G/G609G) by reproducing the same aberrant splicing mechanism leading to progerin production 30. Additionally, we are involved in a consortium with the mouse clinical institute and the medical genetics team in Bordeaux, to explore a KI model of CS carrying the most common H-RAS p.G12S mutation, based on a project funded by the ANR (Cf. Appendix 7).
c. Identify disease mechanisms in progeroid syndromes
d. Establish preclinical proofs of principle for therapeutic approaches and launch clinical trials
Importantly, we recently identified that a specific class of proteasome inhibitors, including MG132, are able to reduce progerin levels by inducing both its degradation through autophagy and inhibition of its production by mRNA splicing modulation45. These findings pave the way for the development of a novel promising class of drugs for the treatment of patients affected with Hutchinson-Gilford Progeria and related syndromes. They may as well find larger applications in diseases involving the accumulation of prenylated proteins or natural aging. This discovery was patented and is co-exploited by ProGeLife (co-owner) and SATT-Sud Est (SATT-SE).
In the age of Assisted Reproductive Technology (ART), where defective gametogenesis is no longer a barrier to procreation, it is crucial to understand how the genome is normally processed in germ cells, detect cases where processing is abnormal and determine the consequences of anomalies for genome stability. We will thus characterize the nuclear lamina during human spermatogenesis, and discover proteins required for nuclear remodelling by searching for genetic causes of abnormal sperm head development (teratozoospermia) and the creation of specific knockout mouse models. Our group has a strong clinical component and enjoys close collaborations with La Conception and La Timone Hospital sites: the Reproductive Biology Laboratory, Centre for the study and conservation of human oocytes and sperm (CECOS, directed by C. Metzler-Guillemain), the Molecular Genetics Laboratory in the Department of Medical Genetics and the Germethèque, a labelled Biobank. We also have a collection of normal testicular material, from patients with brain death, deposited in the Germethèque Biobank and authorized by the Biomedicine Agency (ABM).
The cancer project focuses on the identification of genetic variants that contribute either to increase the risk of digestive cancer or to aggravate their evolution, according to 2 main axes:
A third axis "genetics of sarcomas and related tumors" was initiated this year
Joining Nicolas Lévy's team, the research projects have been re-centered on tumors impairing pathways involved in premature aging, mainly RAS and TGFB, to identify potential therapeutic targets useful in in both conditions. The pancreatic cancerogenesis of mucinous cysts has been chosen as study model.
The projects of the group focused on the study of gastrointestinal tumorigenesis mechanisms to identify genetic variations or mutations that contribute to or increase their risk, or impact their evolution. The clinical objective was to better integrate into medical practice DNA analyses, which take a special place in various aspects of oncology, prevention, early diagnosis and therapeutic evaluation. In the past, we described the genetic alterations responsible for major genetic predispositions to digestive cancer and their relationship with the main phenotypic characteristics. The molecular analyses have been progressively transferred to hospital laboratories and integrated to the medical care of patients. We coordinate the data collection of germ line mutations identified in the French labeled laboratories in national mutations databases and permanently implement these LSDBs with functional data allowing variants classification. Furthermore, the study of somatic genetic alterations in colorectal cancer, to which we contributed, identified two alternative pathways of colon cancer, linked to different prognostic risks. The allelic loss frequencies and the complexity of their associations, as well as the difficulty of finding a major event in tumor progression prompted us to undertake a comprehensive genomic characterization of colorectal cancer combining genome and transcriptome analysis. The direct responsibility of the WNT pathway via the APC gene appeared the most probable hypothesis that was specifically explored 59. To document the existence of genes predisposing to the development of metastases based on genotype-phenotype associations, we set up a national group of clinicians and researchers to collect such case-control series and were able to describe the first locus involved in the metastatic process 60, where a non-coding RNA was then identified by another group 61, 62.
An interface contract with the Institut Paoli-Calmettes allowed us to develop a regional laboratory that contributes to the diagnostic and therapeutic evaluation of cancer.
Joining Nicolas Lévy's team, the group will re-direct its research projects on tumors impairing pathways also involved in premature aging, mainly RAS and TGFB, to identify potential therapeutic targets in pathways involved in both conditions. Since 2006, targeted therapies have been developed to improve solid tumors treatment, like anti-EGFR and Tyrosine kinase inhibitors that are now used in metastatic colorectal and lung cancers. Anti-EGFRs have been first demonstrated as ineffective in case of K-RAS gene activation, and their use has been restricted to tumors without K-RAS somatic mutations. Subsequently, a systematic molecular analysis of tumor cells has been introduced in cancer patients’ management, and the EGFR pathway has been more extensively characterized to document the mechanisms of drug resistance. Similar observations have been made in cases of N-RAS, BRAF or PTEN mutations as examples. Drugs targeting other steps of the pathway have been tested and the use of prenylation inhibitors revealed promising directions to manage devastating tumors such pancreatic, lung and ovarian cancers 63, 64 for reviews. The use of xanthohumol, a prenylated chalcone, is able to induce cell cycle arrest and apoptosis by inhibiting phosphorylation of STAT3, together with expression of downstream targets cyclin D1 and survivin in cell lines and xenografts of pancreatic cancer 65. Combined geranylgeranyltransferase 1 and farnesyltransferase inhibitors liposomal delivery induces G1 cycle cell arrest by p21CIP1/WAF1 in human pancreatic and lung cancer cell lines 66. Zoledronic acid on lung cancer and melanoma cells with NRAS mutation inhibited prenylation, supressing downstream RAS and EGFR signaling. This effect has been reversed using geranylgeraniol and farnesol, confirming the specific prenylation inhibition 67, 68. In combination with paclitaxel, which showed promising results in pancreatic cancer, synergistic effects could be obtained. Blocking the mevalonate pathway with statins also shows interference with post-translational modification of KRAS inducing antiproliferative phenotype of ovarian cancer cells from xenografts 69, suppression of proliferation linked to endocrine resistance resulting from the accumulation of senescent cells after radio/chemotherapy of breast and prostate cancer 70, 71. Isoprenoid biosynthetic pathway inhibitors also showed Rap1A alteration in metastatic prostate cancer cells 72.
Pancreatic cancer is one of the most lethal human cancers, exhibiting RAS pathway activation in more than 80% of cases. The unique treatment consists in chemotherapy based on 5FU or gemcitabine solely or in combination with irinotecan and/or oxaliplatine, adjuvant to surgery in less than 20% of cases, often used as palliative. Clinical trials currently introduce other molecules without any positive result. Pancreatic cancer also takes part of the tumor spectrum of several genetic disorders highly predisposing to cancer (BRCA, Lynch, Peutz-Jeghers, Cowden, Von Hippel Lindau syndromes as the most frequent). Precancerous lesions have been described, particularly pancreatic cysts with mucinous component, known at high risk for cancer. These tumors were retained as an interesting model to extensively study the EGFR/RAS pathway, looking for targetable modifications. A collaborative consortium has been set up in Marseille, including all interventional digestive endoscopy units and the corresponding surgical departments (AP-HM hospitals Timone and Nord, St-Joseph hospital, European Hospital, Institut Paoli-Calmettes), and two expert centers in Lyon (Mermoz hospital) and Paris (Les Peupliers hospital). We plan to collect samples of cystic fluid and analyze“circulating” DNA from surrounding epithelial cells. As blood circulating tumor cells showed their ability to reveal molecular alterations as cancer diagnostics or recurrence markers, tumor cells are initially present in cystic fluids and can be collected for further molecular investigations 73, 74.
A pilot study has just been funded, which will test the feasibility of a systematic approach comparing the molecular profiles of cysts in pre-operative echoendoscopic biopsies and per-operative samples of 10 patients, thus validating the preoperative approach as applicable to a larger series, as echoendoscopic biopsies are systematically performed before surgery to confirm the clinical indication of pancreatectomy. Sample processing and NGS sequencing will be done within U910. Nucleic acids will be extracted and sequenced on a panel of 70 genes involved in pancreatic tumorigenesis and targeting the RAS signaling pathway, MAPK, AKT, JAK-STAT signaling pathway, WNT, TGFB, TP53 and repair BRCA, ATM. The selected sequencing technology (HaloPlexHS®, Agilent), through the incorporation of random nucleic acid sequences (10 nucleotides) during the libraries preparation, enables detection of alleles present in a 1% proportion. This new technical approach that combines high sensitivity and specificity is also compatible with degraded DNA and/or small amounts (less than 50 ng). It therefore appears as an appropriate technique for this type of samples with an expected important degradation and nucleic clonal heterogeneity. Bioinformatics analysis will detect somatic variations and define their allelic frequency as well as CNVs. A comparison of the molecular profiles of pre- and post-operative samples will be performed, then faced with the pathologic and biological characteristics. The identification of a molecular profile indicating a precancerous condition would be helpful for the surgical decision, which consists in a (cephalic) pancreatectomy, now performed twice more than a posteriori required. The characterization of the very early recurrent molecular alterations would also be helpful to refine targeted therapies, and to test cell models exhibiting similar pathways disorders.
At the same time, the group, in collaboration with French clinical oncogenetics units, is currently collecting families with aggregation of breast/pancreatic cancer without known germline alterations to define through exomes analyses a set of candidate genes that could be involved in such predispositions. Variations identified by this approach will be compared to those enlightened in the previous part, to document the genetic alterations responsible for the very early steps of pancreatic tumorigenesis.
Finally, as multichemotherapy is currently the single therapeutic approach in pancreatic cancer, the group drives pharmacogenetics studies to possibly add new parameters useful in dosage adjustment in collaboration with the oncology units of the university hospital. This year, clinical files of 98 patients have been registered and 87 have been genotyped on 1636 SNPs of 231 genes using the DMET chip (Affymetrix). Associations with toxicity have been identified for genes participating to phases I and II metabolism enzymes and transporters. The set of data will be extended, genotyping 279 patients included in the national clinical trial PRODIGE35 in collaboration with the Fédération Française de Cancérologie Digestive (FFCD) to validate primary results.