Ph.D Christian Bailly
email: christian.bailly@pierre-fabre.com

Ready for a comeback of natural products in oncology

Keywords: Cancer, natural products, targets CRC, colorectal cancer, FDA, Food & Drug Administration, high throughput screening, HTS, NP, natural products, NSCLC, non small cell lung cancer, SAR, structure-activity relationship

Since the late 1990s and the rapid expansion of monoclonal antibodies and synthetic protein kinase inhibitors in oncology, anticancer natural products fell out of fashion with the pharmaceutical industry. But in 2007 with the approval of three new drugs derived from natural products, the emergence of promising antitumor compounds from microorganisms (e.g. alvespimycin, salinosporamide) and the growing importance of new formulations of known natural product-derived drugs (nanoparticle formulations, oral forms), we are witnessing a new wave for natural products in oncology. The recent approval of the microtubule-targeted epothilone derivative ixabepilone (Ixempra ® ), the DNA-alkylating marine alkaloid trabectedin (Yondelis ® ) and the inhibitor of mTOR protein kinase temsirolimus (Torisel ® ) is emblematic of the evolution of the field which combines the long established finding of conventional cytotoxic agents and the emergence of molecularly targeted therapeutics. These three examples also illustrate the increasing importance of microbial sources for the discovery of medically useful natural products. The contribution of innovative biological targets is also highlighted here, with references to proteasome inhibitors and novel approaches such as manipulation of mRNA splicing. Altogether, these observations plead for the return of natural products in oncology.

Introduction

The space race and the war on cancer both begun in the 1950s. When Yuri Gagarin first went into orbit around the earth in 1961, key antitumor antibiotics like doxorubicin and mitomycin were discovered. When Neil Amstrong put his foot on the moon on July 20 1969, some novel antitumor agents such as bleomycin and vincristine were just launched. Over the past 50 years, natural products (NP, here the term is restricted to small molecules) have been the cornerstone of anticancer pharmacology. The discovery of these antitumor NP opened the route to the "forty glory cytotoxic", 4 decades of cytotoxic agents, among which a significant number of molecules from plants and microorganims still used today for the treatment of cancer (Fig. 1). Many of these old drugs remain largely prescribed and have saved or prolonged the life of millions of patients with cancer. But at the end of the 1990s, a novel onco-pharmacological universe was discovered, with the use of targeted therapeutics made of receptor-specific small (imatinib) or large (rituximab) molecules. Immediately, the antitumor pharmacology community embraced the new concept and this vision of molecular targetbased drug discovery (or reverse pharmacology) became a standard. At this stage, the search for natural products in oncology declined and there was hope to envisage rapidly a personalised treatment of cancer with rationally designed magic bullets, silico-or omicocontrolled. The objective remains today but the route is long and progresses are slow. There is no doubt that the treatment of cancers has profoundly changed, with the advances of targeted therapies. But major differences remain between the different types of tumors. Patients suffering from chronic myeloid leukaemia have a good probability of long-term survival, thanks to the discovery of specific tyrosine kinase inhibitors like imatinib (Gleevec ® ), dasatinib (Sprycel ® ) or nilotinib (Tasigna ® ). In sharp contrast, the therapeutic options for patients with advanced melanoma remain quite limited and the survival prognostic is still extremely low. The activity of dacarbazine (DTIC) against metastatic melanoma was first noticed in the early 1970s [START_REF] Carter | 5-(3,3-dimethyl-l-triazeno)-imidazole-4-carboxamide (DTIC, DIC, NSC-45388)--a new antitumor agent with activity against malignant melanoma[END_REF] and 36 years later, this product remains a standard: where is the progress? For the majority of solid tumors, in particular for advanced and metastatic grades, treatment modalities remain often palliative and very insufficient, despite the development of newer drugs supposed to provide novel therapeutic options. These drugs are effective, otherwise they would not be on the market, but in many cases, they only prolong the life of patients for several weeks or a few months, rarely for years. There is a strong need for novel anticancer drugs effective against solid tumors, especially at advanced stages of the disease.

The need is large and urgent for nearly all solid tumors. Just one example: every 3.5 minutes, A c c e p t e d M a n u s c r i p t 4 someone is diagnosed with colorectal cancer (CRC); every 9 minutes, someone dies from CRC; and every 5 seconds, someone who should be screened for CRC is not [START_REF] Benson | Epidemiology, disease progression, and economic burden of colorectal cancer[END_REF]. We definitely need new drugs, as well as new combinations, new strategies to select patients and to evaluate treatment responses. As regards drugs, I believe that NP should continue to be considered as one of the most important sources of innovative products. Astronauts apparently plan to go back to the moon in the near future, possibly 2011, to learn new things, develop innovative research programs and demonstrate that they can essentially "live off the land". By analogy, a new vision for NP space exploration can be envisioned to drive drug discovery and spur new medical advancements, especially in the field of cancer through the exploration of novel biodiversity sources and emerging targets. In this commentary, I tried to analyze the current situation with the place and role of NP in modern oncology, discuss about recent evolutions and some perspectives. This is not an exhaustive survey of NP in cancer, there are excellent recent reviews for that [START_REF] Butler | Natural products to drugs: natural product-derived compounds in clinical trials[END_REF][START_REF] Butler | Mother Nature's gifts to diseases of man: the impact of natural products on anti-infective, anticholestemics and anticancer drug discovery[END_REF] but essentially a personal vision of the field at the interface between chemistry and biology.

An excellent track record for natural products in oncology

Old good drugs from plants and microbes remain essential. Since the 1950s, a large number of novel antitumor drugs have been identified and validated clinically (Fig. 1). However, the proportion of NP is certainly decreasing, mainly due to the advance of small synthetic molecules (in particular kinase inhibitors) and the fast growing development of monoclonal antibodies. Nevertheless, NP remain essential and novel molecules of marine or terrestrial origins are regularly approved for the treatment of cancers. Examples of recently launched NP and derivatives will be discussed below. Known molecules too continue to contribute considerably to cancer chemotherapy. The anthracyclines such as doxorubicin, epirubicin and daunorubicin cannot be ignored. This is especially the case for doxorubicin (Adriamycin ® ) extensively used for the treatment of breast cancer, despite the risk of cardiotoxicity particularly in patients with underlying cardiac issues or the elderly. This drug is also frequently associated with monoclonal antibodies, with trastuzumab for breast cancers, with rituximab for lymphoma, for examples. There are numerous chemotherapy regimens associating doxorubicin with chemo-and bio-therapeutics. In the early seventies there was initial clinical reports on the use of doxorubicin in the treatment of Hodgkin disease, leukaemia, breast, lung and cervical cancers. In one way, it is regrettable to admit that a quarter of century later, this bacterial product qualified in 1974 as a new drug with significant Vinorelbine (Navelbine ® ), arguably the major semi-synthetic tubulin-binding vinca alkaloid, first approved in Europe in 1989 and in the USA in 1994 for the treatment of non-small cell lung cancer (NSCLC) then of advanced breast cancer, remains today extensively used worldwide. Similarly, paclitaxel (Taxol ® ) and its rival docetaxel (Taxotere ® ) continue to dominate the cytotoxic market.

New formulations of known natural products

New forms of known natural products are being continuously developed to facilitate the handling of the drugs (for example for poorly soluble compounds) or limit their side toxicities. This is typically the case for the two taxanes paclitaxel and docetaxel, both effective but which often encounter undesirable side effects as well as drug resistance. Second generation taxanes are being developed such as ortataxel or more recently TPI 287 (Tapestry Pharmaceuticals) engineered to overcome multi-drug resistance and to bind to mutant tubulin.

Taxane-containing polymers are also investigated such as paclitaxel poliglumex (Opaxio ® formerly known as Xotax ® ) or Taxoprexin ® made by linking paclitaxel to the natural fatty acid DHA (docosahexaenoic acid). The leading compound has been ABI-007 (Abraxane ® from Abraxis Bioscience), approved by the U.S. Food & Drug Administration (FDA) in January 2007 for the treatment of metastatic breast cancer and which is an albumin-stabilized, nanoparticle formulation of paclitaxel for injectable suspension. This form was found to have significantly less toxicity than Cremophor-containing paclitaxel in mice [START_REF] Gradishar | Clinical efficacy and emerging therapeutic utilization of novel taxanes[END_REF][START_REF] Aapro | Molecular basis for the development of novel taxanes in the treatment of metastatic breast cancer[END_REF]. Solvent-based formulations of taxanes are commonly associated with hypersensitivity reactions, neutropenia, and neuropathy. Patients with metastatic breast cancer treated with this nanoparticule formulation (mean particle size of about 130 nm) achieved a significantly higher objective A c c e p t e d M a n u s c r i p t 6 response rate and time to progression than those treated with Cremophor-containing paclitaxel. This nab-paclitaxel form may therefore enable clinicians to administer paclitaxel at higher doses with less toxicity than is seen with Cremophor-containing paclitaxel [START_REF] Scripture | Paclitaxel chemotherapy: from empiricism to a mechanism-based formulation strategy[END_REF]. Similar albumin-based drug development are ongoing with other water-insoluble anticancer agents such as docetaxel and rapamycin [START_REF] Hawkins | Protein nanoparticles as drug carriers in clinical medicine[END_REF][START_REF] Wong | Suspensions for intravenous (IV) injection: A review of development, preclinical and clinical aspects[END_REF]. A similar trend can be evoked with the camptothecin-derived topoisomerase I inhibitor topotecan (Hycamtin ® ) initially approved in 1996 for the treatment of small cell lung cancer (SCLC) after failure of first-line chemotherapy and metastatic ovarian carcinoma after failure of initial or subsequent chemotherapy. Since the approval of injectable topotecan for the second-line treatment of SCLC, new formulations have been developed. In October 2007, more than 10 years later, a new oral formulation of the drug as capsules for the treatment of patients with relapsed small cell lung cancer was proposed [START_REF] Nicum | Topotecan for the treatment of small-cell lung cancer[END_REF]. Oral topotecan will represent a good candidate for combination therapy with other i.v. or oral chemotherapy agents, monoclonal antibodies, and small molecule tyrosine kinase inhibitors. In the same vein, oral availability and administration of some of the newer CPT analogues, including diflomotecan (BN80915) and gimatecan (ST1481), have also shown promising results.

One of the most typical example of reformulation is that of pegylated liposomal doxorubicin (Doxil ® from Tibotec Therapeutics; Caelyx ® from Schering-Plough) which has demonstrated efficacy in the treatment of recurrent ovarian cancer. The liposome-encapsulated form improves doxorubicin penetration into tumors and decreases drug clearance, thereby increasing the duration of the therapeutic effect. Pharmacokinetic studies have indeed revealed that the liposomal form results in a longer half-life with less free drug available for tissue distribution than conventional doxorubicin. This liposomal formulation of doxorubicin reduces toxicity, specifically the cardiac effects commonly seen with anthracycline antitumor drugs but tolerability is often non uniform among patients. Very recently, a liposomeencapsulated cytarabine/daunorubicin combinations with a molar ratio of 5:1, designated CPX-351 (from Celator Pharmaceuticals), has been granted orphan drug designation by the FDA for the treatment of acute myeloid leukemia. Liposomal formulations of platinum drugs are also studied.

Another practical approach to improve the efficacy or facilitate the handling and tolerability (and patients' preferences) of a natural product consists to develop an oral regimen. Most cytotoxics are administrated intravenously, but some are also available in an oral formulation. This is the case for oral vinorelbine formulated in a soft gelatin capsule, with a predictable A c c e p t e d M a n u s c r i p t 7 bioavailability of about 40% [START_REF] Marty | Oral vinorelbine pharmacokinetics and absolute bioavailability study in patients with solid tumors[END_REF] and comparable efficacy to the i.v. form with respect to response rate, progression-free survival and overall survival [START_REF] Jassem | A multicenter randomized phase II study of oral vs. intravenous vinorelbine in advanced non-smallcell lung cancer patients[END_REF]. This orally active vinca alkaloid (Fig. 2) offers novel opportunities for cancer treatment, both in NSCLC and in metastatic breast cancer. Oral formulations can reduce the incidence of side effects associated with an i.v. line (extravasation, pain, local infection, thrombosis), contribute to resource sparing for the health care system and for the patient [START_REF] Aisner | Overview of the changing paradigm in cancer treatment: oral chemotherapy[END_REF] and to alleviating the burden on his/her relatives. Even when combined to intravenous treatments, the oral formulation of vinorelbine is preferred to its intravenous counterpart by 74% of patients [16]. By reducing constraints related to treatment administration, oral agents improve the global efficiency of cancer treatment. Their use is increasing in oncology [17].

Novel approved NP drugs and promising clinical candidates

As far as I know, no novel category of NP was approved by the FDA for the treatment of cancer during years 1997-2006 (Fig. 3). In the same time, resources attributed to NP research in oncology by most major pharmaceutical companies were reduced. During that period, efforts were essentially focused on biotherapies and targeted synthetic small molecules.

However, the field of NP remained active with the clinical development of a few innovative molecules, among which 3 key products, ixabepilone, temsirolimus and trabectedin now registered in the USA and/or in the EU. They are briefly mentioned here:

-Ixabepilone (BMS-247550, Ixempra ® from Bristol-Myers-Squibb). Drugs that target microtubules, including vinca alkaloids (vinorelbine, vincristine) and taxanes (paclitaxel and docetaxel), are arguably the most commonly prescribed anticancer therapies. The use of these "old good drugs" may however be limited by side toxicities and/or resistance induced by efflux proteins. Novel antimicrotubule agents, better tolerated and active in the setting of drug resistance have been searched. This is the context which lead to the discovery of the macrolide antibiotics epothilones, produced by the myxobacterium Sorangium cellulosum, as a novel class of microtubule-stabilizing agents discovered in 1995 [START_REF] Bollag | Epothilones, a new class of microtubule-stabilizing agents with a taxollike mechanism of action[END_REF]. Epothilones A-B exhibit potent in vitro anticancer activity, against taxane-resistant cell lines, but their in vivo activity is modest, owing to a poor metabolic stability and unfavorable pharmacokinetics. The synthesis and testing of more than 300 semi-synthetic epothilone analogues led to the rational design of ixabepilone, which displays reduced susceptibility to a range of common tumor resistance mechanisms. This novel antimicrotubule agent acts in a similar manner to taxanes, approved in the EU as a second-line treatment for advanced soft tissue sarcoma in patients who have failed on anthracyclines and ifosfamide, or who are unsuited to receive these agents. It also has orphan drug status in soft tissue sarcoma in the US and in ovarian cancer in the US and EU, and is under investigation as combination therapy in patients with recurrent ovarian cancer [START_REF] Carter | Trabectedin : a review of its use in the management of soft tissue sarcoma and ovarian cancer[END_REF]. Hopefully, trabectedin has opened up the way to the successful development of other marine NP. There are several marine drug candidates (derived from dolastatin 10, bryostatin 1, didemnin B, kahalalide F, halichondrin B, etc) currently in clinical trials [START_REF] Simmons | Marine natural products as anticancer drugs[END_REF][START_REF] Singh | Biodiversity, chemical diversity and drug discovery[END_REF]. The development of marine toxins remains a difficult challenge but hopefully the next decade will offer a prominent place to sea products [START_REF] Mayer | Marine pharmacology in 2005-2006: Antitumour and cytotoxic compounds[END_REF].

-Temsirolimus (CCI-779, Torisel ® from Wyeth) is the first inhibitor of the protein kinase mTOR (mammalian target of rapamycin) approved for the treatment of patients with advanced renal cell carcinoma [START_REF] Ma | Temsirolimus[END_REF]. The mTOR signaling pathway is recognized as an important mediator in cell proliferation, growth, survival, and angiogenesis. It is the Everolimus is an antiproliferative agent primarily directed at cancer cells, but it is useful in other therapeutic domains as well, in particular for patients with coronary artery disease. A cobalt-chromium stent eluting everolimus was found to be more effective than a paclitaxeleluting stent, with fewer major adverse cardiac events [START_REF] Stone | Comparison of an everolimus-eluting stent and a paclitaxel-eluting stent A c c e p t e d M a n u s c r i p t 21 in patients with coronary artery disease: a randomized trial[END_REF].

From the chemical point of view, these "rolimus" compounds (rapalogs) all derive from rapamycin, the macrolide antibiotic produced by Streptomyces hygroscopicus, initially discovered as an antifungal agent [START_REF] Vezina | Rapamycin (AY 22,989), a new antifungal antibiotic. I. Taxonomy of the producing streptomycete and isolation of the active principle[END_REF] and endowed with anti-inflammatory, anti-tumor and immunosuppressive properties. This Streptomyces strain originates from a soil sample collected in Easter island (Rapa Nui). Poorly soluble in water, sirolimus is given orally whereas temsirolimus is a water-soluble dihydroxymethyl propionic acid ester prodrug mainly used as an iv formulation. It quickly undergoes hydrolysis to sirolimus after iv administration, so that most of its clinical effects are likely attributable to the sirolimus metabolite [START_REF] Hartford | Rapamycin: something old, something new, sometimes borrowed and now renewed[END_REF].

These three examples illustrate the continued successful development of NP in oncology, prolonging thus the long history of NP-based anticancer drug discovery initiated more than 50 years ago. In each case, NP medicinal chemistry has been essential in producing desirable pharmacological properties (such as solubility, distribution or resistance to metabolism…) and structure-activity relationship (SAR) studies have been instrumental to guide drug design and to help comprehend their mechanism of action. In some cases, the unmodified NP itself can become the final product but even in this case, a robust chemistry is needed to synthesize the compound from another precursor (as in the case of trabectedin and cyanosafracin B) and/or to establish SAR. In most cases modifications of the original NP are needed, usually rather A c c e p t e d M a n u s c r i p t 10 simple transformations of the NP to optimize formulation and ADMET properties [START_REF] Ganesan | The impact of natural products upon modern drug discovery[END_REF].

Several other promising NP drug candidates are in the pipeline of clinical trials. These include, to cite only 3 examples, (i) vinflunine (Javlor ® from Pierre Fabre), the latest hemisynthetic vinca alkaloid in development, prepared via super-acidic chemistry and targeting microtubules dynamics, which has revealed promising activity for the treatment of bladder, breast and non-small cell lung cancers [START_REF] Fahy | Vinflunine: discovery and synthesis of a novel microtubule inhibitor[END_REF][START_REF] Bellmunt | Clinical activity of vinflunine in transitional cell carcinoma of the urothelium and other solid tumors[END_REF], (ii) amrubicin (from Pharmion), a thirdgeneration synthetic anthracycline targeting topoisomerase II, which recently received FDA fast track designation for the treatment of SCLC after first-line chemotherapy [START_REF] Kurata | Amrubicin for non-small-cell lung cancer and small-cell lung cancer[END_REF], (iii) alvespimycin (KOS-1022 from Kosan), an orally active heat shock protein (Hsp) 90 inhibitor derived from 17-allylaminogeldanamycin (17-AAG). The Hsp90 multichaperone complex has important roles in the development and progression of malignant transformation, promoting thus the development of Hsp90 inhibitors for cancer treatment [START_REF] Pearl | The Hsp90 molecular chaperone: an open and shut case for treatment[END_REF].

In addition to pure compounds, NP extracts should also be considered. At this level, it is worth to mention the antitumor activity of Kanglaite injection (KLT, from Zhejiang Klanglaite Pharmaceutical Co., China), a micro-emulsion prepared from an extract of the Chinese plant Semen coicis [START_REF] Lu | Chinese herb related molecules of cancer-cell-apoptosis: a minireview of progress between Kanglaite injection and related genes[END_REF]. In 1997, a phase III clinical study was successfully completed and the injection product was officially launched in China. KLT was later approved by the Russian Federation where it is now marketed. Kanglaite was the first drug derived from a traditional Chinese herbal remedy to go into clinical trials in the USA [START_REF] Normile | Asian medicine: the new face of traditional Chinese medicine[END_REF].

Microorganisms: the main supplier of anticancer structurally novel NP ?

For the past 40 years, plants have been a great source of secondary metabolites with extreme chemical diversity. A number of plant-derived anticancer drugs belonging to camptothecins, taxanes, vinca alkaloids and the epipodophyllotoxins families, remain extensively prescribed.

But beside these well-known families, the number of plant-derived drug candidates that have reached clinical development in oncology has been reduced since 2000 (Fig. 3). Different pharmaceutical companies carry on with the development of plant NP, for examples Unibioscreen with the 2''-oxovoruscharin derivative UNBS1450 hemisynthesised from a cardenolide extracted from Calotropis procera [START_REF] Mijatovic | Nucleolus and c-Myc: potential targets of cardenolide-mediated antitumor activity[END_REF], Pierre Fabre with a hemisynthetic triptolide derivative from the Chinese medicinal plant Tripterygium wilfordii or Oxigene with the microtubule-depolymerizing vascular disrupting agent combretastatin-A4 phosphate (Zybrestat ® , [START_REF] Petit | The microtubule-targeting agent CA4P regresses leukemic xenografts by disrupting interaction with vascular cells and mitochondrialdependent cell death[END_REF]). But overall plants now receive less attention from pharmaceutical companies, perhaps because of a (real or perceived) reduced chemo-diversity. But it is A c c e p t e d M a n u s c r i p t 11 estimated that less than 15% of higher plants have been systematically investigated for the presence of bioactive compounds [START_REF] Pieters | Bioguided isolation of pharmacologically active plant components, still a valuable strategy for the finding of new lead compounds?[END_REF]. Therefore, there is certainly room to explore further the plant kingdom, more deeply and perhaps differently compared to what has been done so far, using novel targets and novel methodological approaches. Despite these difficulties, several authors maintain the idea that plant NP can return to the forefront of anticancer pharmacology [START_REF] Mcchesney | Plant natural products: back to the future or into extinction?[END_REF]. In the past, traditional screening approaches have allowed a rich harvest of "low hanging fruits". Additional efforts should be devoted now to reach for the highhanging fruits, with the help of new techniques and innovative targets.

Marine macroorganisms, another great source of secondary metabolites, have also lost some popularity in the pharmaceutical industry in recent years, mainly due to the difficulty of large scale supply. Certain companies remain very active in this domain, such as PharmaMar (Madrid, Spain) with an impressive pipeline of marine anticancer agents in development (aplidin, kahalalide F, Zalypsis,…), but the current tendency is clearly toward the exploitation of microbial sources of NP. Marine macroorganisms are difficult to collect in large scale. Moreover, it is frequent that the compound of interest initially found in the macroorganism derives in fact from a symbiotic microorganism. A typical example is that of the dolastatins first isolated from the mollusc Dolabella auricularia and later found to be produced by cyanobacteria [START_REF] Sielaff | Natural products from cyanobacteria: Exploiting a new source for drug discovery[END_REF]. This is also the case for ecteinascidin as stated above, and most likely for discodermolide, halichondrin B and bryostatin 1. A significant portion of the bioactive metabolites thought originally to be products of the source animal are often synthesized by their symbiotic microbiota [START_REF] Simmons | Biosynthetic origin of natural products isolated from marine microorganism-invertebrate assemblages[END_REF]. Microorganisms are the most attractive source for the production of medically useful secondary metabolites. Many microorganisms can be grown to large scales in culture media, facilitating an unlimited and uninterrupted supply of the raw material needed for drug development. Actinomycetes have been one of the most investigated group of bacteria during the past 50 years. Filamentous fungi have been largely studied too.

But even if these two groups have already received a major attention, there is still room for the discovery of highly potent anticancer NP in these families. It is estimated that the number of antibiotics characterized to date represents less than 5% of the total. In other words, the chemical universe produced by actinomycetes remains largely underexploited [START_REF] Singh | Biodiversity, chemical diversity and drug discovery[END_REF]. In the next ten years, this percentage should be increased significantly with the improvement of cultivation methods of prokaryote organisms and potentially the growing exploitation of metagenomic approaches for uncultivable microorganisms. New compounds belonging to novel structural classes and possessing unprecedented biological properties can be discovered A c c e p t e d M a n u s c r i p t 12 from microorganisms, whereas this is perhaps less probable with plants. In addition, the improvement of cultivation techniques together with the refinement of highly sensitive analytical methods should help the rapid dereplication of compounds [START_REF] Lang | Evolving trends in the dereplication of natural product extracts: new methodology for rapid, small-scale investigation of natural product extracts[END_REF][START_REF] Koehn | High impact technologies for natural products screening[END_REF]. In the near future, it is likely that the exploration of ecological niches and habitats, in particular marine environments with a large biodiversity, will furnish structurally novel NP of prime pharmacological interest. Personally, I share the view that a renaissance of drug discovery inspired by natural products can be anticipated for the next decade [START_REF] Harvey | Natural products as a screening resource[END_REF]. NP may not totally recapture the leading position they once held as a foundation for anticancer drug discovery and development but they will continue to play a major role in antitumor pharmacology and drug discovery. We already see the premises of the renaissance of NP with the approval of the aforementioned anticancer drugs ixabepilone, trabectedin and temsirolimus, and with other bacterial products such as the histone deacetylase inhibitor depsipeptide Romidepsin (FK228) which has received a fast-track status by the FDA in October 2004 as monotherapy for the treatment of patients with refractory cutaneous T-cell lymphoma, i.e. only ten years after its first isolation from Chromobacterium violaceum No.968 [START_REF] Masuoka | Histone deacetylase inhibitors from microorganisms: the Astellas experience[END_REF]. Even if the discovery of tumoractive NP remains a critical challenge and a risky option in the pharmaceutical industry, this strategy does not necessarily take longer time than chemistry-based small chemical molecules drug discovery.

Natural product-biological target: a pas de deux in drug discovery

In a pas de deux, the French for "step of two" in a ballet, the man plays a pivotal role by stabilizing, lifting, and turning the lady to take positions she would never be able to do on her own. A close complicity between the two partners is essential. A parallel situation occurs in drug discovery strategies where biological targets and NP-based libraries must be adapted to maximize their exploitation. NP drug discovery is guided at every turn by biological assays.

Chances of discovering products of pharmacological interest depend on the quality of the NP source with a large biodiversity, adapted cultivation processes, resources of chemistry, etc. but it depends also on the quality of the targets or biological procedures set up to detect the compounds of interest. Not all molecular targets are well adapted to the identification of pharmacologically active NP. For example, the screening of natural extracts against purified protein kinases is generally difficult, with a higher percentage of false positives compared to synthetic chemical libraries. Enzyme-linked immunosorbent assays and cell-based assays are certainly better adapted to the screening of NP extracts. The design of highly robust and A c c e p t e d M a n u s c r i p t 13 sensitive high throughput screening (HTS) assays is a key element. For example, the specific design of a HTS procedure for the discovery of naturally-occurring proteasome inhibitors [START_REF] Ausseil | High-throughput bioluminescence screening of ubiquitinproteasome pathway inhibitors from chemical and natural sources[END_REF] allowed us to identify original molecules from a plants collection, such as physalin A from the tropical herb Physalis angulata [START_REF] Vandenberghe | Physalin B, a novel inhibitor of the ubiquitin-proteasome pathway, triggers NOXA-associated apoptosis[END_REF].

The most popular approach to identify novel NP in oncology refers to cell cytotoxicity measurements. Cell-based assays are routinely used to evaluate the cytotoxicity of standardised extracts (or pure natural products) against murine or human cancer cells cultivated in vitro. Extract A tested against cell type B leads to the isolation of a novel cytotoxic agent C is a typical published story. The cell type can be varied almost indefinitely to include cancer cells of different tissue origin (breast, lung, prostate, bladder…), cell lines sensitive or resistant to a given anticancer drug or even multi-resistant, cells deficient for a specific enzyme (e.g. topoisomerase-mutated cells) or an altered pathway (e.g. cell with deficient DNA-repair), the use of non-tumoral (but often immortalised) "normal" cells, etc. These models are useful, robust and easy to set up but often too permissive. These monolayer culture models are routinely employed to identify conventional cytotoxic agents which then have to be tested in animal models, requiring long and expensive procedures. Whenever possible, this classical approach should be replaced with more sophisticated alternative screening procedures. The use of sophisticated models, more stringent and of a higher predictive value for the clinical efficacy should be favoured. An interesting approach consists to use multicellular tumor spheroid models which are of intermediate complexity between in vivo tumors and in vitro monolayer cultures. Multicellular spheroids can be generated in 96well plates format for HTS, from a variety of tumor cell lineages [START_REF] Ivascu | Rapid generation of single-tumor spheroids for high-throughput cell function and toxicity analysis[END_REF][START_REF] Herrmann | Screening for compounds that induce apoptosis of cancer cells grown as multicellular spheroids[END_REF]. Similar approaches can be established to study heterologous cell interactions in solid tumors and the cellular tumor environment [START_REF] Friedrich | Experimental anti-tumor therapy in 3-D: spheroids-old hat or new challenge?[END_REF]. Such 3-D culture models hold great promise to investigate the role of certain tumor initiating cells and their growth inhibition by specific natural products. It is time now to incorporate these 3-D test systems into drug development operations.

Beside cytotoxicity measurements, screening procedure for the discovery of novel NP frequently relies on the use of specific cell lines adapted or engineered to detect drug-induced apoptosis [START_REF] Tian | A high throughput drug screen based on fluorescence resonance energy transfer (FRET) for anticancer activity of compounds from herbal medicine[END_REF], cell cycle perturbations or alteration of specific molecular pathways. A typical example is that of the proteasome, a key component for limiting tumor cell proliferation and a target particularly well suited to the identification of NP. Proteasome inhibitors induce programmed cell death preferentially in transformed cells [START_REF] Orlowski | Proteasome inhibitors in cancer therapy: lessons from the first decade[END_REF]. The drug bortezomib (Velcade ® from Millenium) has been approved initially for the treatment of A c c e p t e d M a n u s c r i p t 14 relapsed/refractory multiple myeloma and more recently for the treatment of mantle cell lymphoma, for which this agent has become a standard of care. This boronic acid dipeptide is an effective reversible inhibitor of the chymotryptic protease in the 26S proteasome, which blocks activation of nuclear factor κB, resulting in increased apoptosis, decreased angiogenic cytokine production, and inhibition of tumor cell adhesion to stroma [START_REF] Roccaro | Bortezomib in the treatment of cancer[END_REF]. The development of a new generation of proteasome inhibitors that hold the promise of efficacy in bortezomibresistant tumors is an active pharmacological field. Arguably the most promising drug in this category is the chlorinated natural product salinosporamide (Nereus Pharmaceuticals) from the marine actinomycete Salinispora tropica. Unlike bortezomib, which is a reversible inhibitor, salinosporamide covalently binds to the proteasome, resulting in the irreversible inhibition of 20S proteasome activity. Salinosporamide was shown to act synergistically with bortezomib to trigger apoptosis in multiple myeloma cells in vitro and in vivo in a human plasmacytoma xenograft mouse model [START_REF] Chauhan | A novel orally active proteasome inhibitor A c c e p t e d M a n u s c r i p t 23 induces apoptosis in multiple myeloma cells with mechanisms distinct from Bortezomib[END_REF][START_REF] Chauhan | Combination of proteasome inhibitors bortezomib and NPI-0052 trigger in vivo synergistic cytotoxicity in multiple myeloma[END_REF]. This natural product is active orally and has shown efficacy in a range of animal models for both hematological malignancies and solid tumors. It is currently undergoing phase I clinical trials. Salinosporamide, and the structurallyrelated γ-lactams omuralide and lactacystin, also illustrate the interest of exploiting microbial natural products in the search for novel anticancer agents.

Another novel promising irreversible inhibitor of the proteasome is carfilzomib (PR-171, Proteolix Inc.), derived from the actinomycete metabolite epoxomicin, which inhibits proliferation and activates apoptosis in patient-derived multiple myeloma cells. This epoxyketone peptidyl inhibitor shows increased efficacy compared with bortezomib and is active against cell samples from patients with clinical bortezomib resistance [START_REF] Kuhn | Potent activity of carfilzomib, a novel, irreversible inhibitor of the ubiquitin-proteasome pathway, against preclinical models of multiple myeloma[END_REF]. Carfilzomib is currently in Phase 2 clinical trials in patients with relapsed or refractory multiple myeloma and in Phase 1 in lymphoma. Other microbial products targeting the proteasome have been identified, such as the cinnabaramides isolated from a terrestrial streptomycete, belactosines A and C from Streptomyces sp., the cyclic peptide TMC-95 from the fungus Apiospora montagnei, Fellutamide B from Penicillium fellutanum, and syringolin A secreted in plants by a strain of Pseudomonas syringae [START_REF] Moore | Advances in and applications of proteasome inhibitors[END_REF]. Syringolin A is a pseudo-peptide virulence factor that irreversibly inhibits all three catalytic activities of eukaryotic proteasomes, via a novel mechanism of covalent binding to the catalytic subunits [START_REF] Groll | A plant pathogen virulence factor inhibits the eukaryotic proteasome by a novel mechanism[END_REF]. The α,β-unsaturated carbonyl of syringolin A reacts with a threonine residue of the proteasome to form a covalent bond and this cross-linking is considered at the origin of the antiproliferative and pro-apoptotic effects observed in ovarian and neuroblastoma cancer cells [START_REF] Coleman | Syringolin A, a new plant elicitor from the phytopathogenic bacterium Pseudomonas syringae pv. syringae, inhibits the proliferation of neuroblastoma and ovarian cancer cells and induces apoptosis[END_REF]. Proteasome inhibitors have also been identified from plant extracts. This is the case for celastrol from the Chinese medicinal A c c e p t e d M a n u s c r i p t 15 plant Tripterygium wilfordii, withaferin A from the medicinal plant "Indian Winter Cherry", pristimerin from plants of the Celastraceae and Hippocrateaceae families, and physalin A from the plant Physalis angulata, as mentioned above. But of course, this field is not restricted to natural products and promising synthetic molecules have been reported as well, such as the phenylpyridine-containing boronic acid derivative CEP-18770 which is a novel orally-active inhibitor of the chymotrypsin-like activity [START_REF] Dorsey | Discovery of a potent, selective, and orally active proteasome inhibitor for the treatment of cancer[END_REF][START_REF] Piva | CEP-18770: A novel, orally active proteasome inhibitor with a tumor-selective pharmacologic profile competitive with bortezomib[END_REF]. These proteasome inhibitors are primarily designed as anticancer agents but they can be of interest for other pathologies characterized by alterations in the proteasome proteolytic pathway including autoimmune and inflammatory diseases, myocardial infarction, and ischemic brain injury [START_REF] Shah | The ubiquitin-proteasome system and proteasome inhibitors in central nervous system diseases[END_REF]. The use of proteasome inhibitors has been suggested for the treatment of rheumatoid arthritis. In this case, the rational is to limit the growth of fibroblast-like synoviocytes that mediate joint destruction [START_REF] Brun | Proteasome inhibition as a novel therapy in treating rheumatoid arthritis[END_REF].

Many new targets have been discovered and biological/biochemical tools such as sh/siRNA or engineered cellular and animal models are useful to validate these targets. But in parallel, the full potential of old targets should be exploited further. Just to cite one example, topoisomerase I is a well-known target in oncology but thus far there is only one chemical series of inhibitors on the market: the camptothecins (Fig. 4). Topotecan, irinotecan, and newer compounds in clinical trials such as gimatecan and diflomotecan all exploit the same property of the enzyme, DNA cleavage which they promote through stabilization of the DNAtopoisomerase I covalent complex which is normally transient. There is room for more potent, better tolerated and orally active inhibitors acting at this level, such as the indolocarbazole edotecarin, the indenoisoquinoline derivative NSC 724998 and the dibenzonaphthyridinone ARC-111 for examples [START_REF] Teicher | Next generation topoisomerase I inhibitors: Rationale and biomarker strategies[END_REF]. But there are also opportunities to target other activities of this key enzyme for tumor cell proliferation. Topoisomerase I interacts with a large number of specific proteins important for the control of transcription and DNA repair, such as the coactivator of p53 topors, nucleolin, the tumor suppressor proteins prostate apoptosis response-4 (Par-4) and NKX3.1, SV40 large T antigen and certain helicases (Fig. 4). These protein interfaces may be targeted, if suitable assays can be developed. Topoisomerase I also carries a kinase activity important for the regulation of RNA splicing [START_REF] Rossi | Specific phosphorylation of SR proteins by mammalian DNA topoisomerase I[END_REF]. Although the link between the topoI kinase activity and cancer is still poorly understood, it is nevertheless clear that this is a potential new area for pharmacological intervention. In addition to its wellknown DNA relaxation activity, human topoisomerase I is responsible for the phosphorylation of Ser/Arg-rich (SR) splicing proteins, in particular the splicing factor A c c e p t e d M a n u s c r i p t 16 2/alternative splicing factor. SF2/ASF and topoisomerase I regulate their activity via an interaction between two closely-spaced RNA recognition motifs of the splicing factor and the N-terminal cap region of topoisomerase I which is essential for the kinase reaction [START_REF] Trzcińska-Daneluti | RRM proteins interacting with the cap region of topoisomerase I[END_REF]. This mechanism can be modulated with NP and derivatives such the glycosylated indolocarbazole derivative NB-506, derived from the streptomycete metabolite BE-13793C and from rebeccamycin produced by the actinomycete Saccharothrix aerocolonigenes. NB-506 acts as a potent inhibitor of both kinase and relaxing activities of topoisomerase I (Fig. 4), and modulates pre-mRNA splicing through inhibition of SF2/ASF phosphorylation [START_REF] Pilch | Specific inhibition of serine-and arginine-rich splicing factors phosphorylation, spliceosome assembly, and splicing by the antitumor drug NB-506[END_REF]. SF2/ASF is as a splicing factor and also mediates postsplicing activities such as mRNA export and translation initiation of bound mRNAs, through interactions between SF2/ASF with both the mTOR kinase and the phosphatase PP2A. SF2/ASF functions as an adaptor protein to recruit signaling molecules responsible for regulation of translation of specific mRNAs [START_REF] Michlewski | The splicing factor SF2/ASF regulates translation initiation by enhancing phosphorylation of 4E-BP1[END_REF]. The phosphorylation state of the SR domain influences the role of SF2/ASF in cytoplasmic RNA processing. A model has been proposed whereby reversible protein phosphorylation differentially regulates the subcellular localization and activity of shuttling SR proteins [START_REF] Sanford | Reversible phosphorylation differentially affects nuclear and cytoplasmic functions of splicing factor 2/alternative splicing factor[END_REF].

Moreover, recent studies have revealed that SF2/ASF is up-regulated in various human tumors and can act as an oncoprotein [START_REF] Karni | The gene encoding the splicing factor SF2/ASF is a proto-oncogene[END_REF]. Therefore it might be valuable to try to manipulate SF2/ASF-mediated splicing via an interference with the kinase activity of topoisomerase I.

The same approach can be envisaged with other kinases involved in the regulation of SF2/ASF-dependent splicing such as Cdc2-like kinase-1 (Clk-1) [START_REF] Muraki | Manipulation of alternative splicing by a newly developed inhibitor of Clks[END_REF]. There are multiple pharmacological options to modulate the activity of SR splicing factors and to try to control splicing [START_REF] Soret | SR proteins as potential targets for therapy[END_REF]. There is an estimated 100,000 splice variants of messenger RNA transcripts from the approximately 25,000 genes estimated in humans. This area of pharmacology is practically a desert.

Novel targets including isolated molecular receptors and more complex phenotypic signals, and old targets revisited offer multiple perspectives to exploit differently NP and bio-extracts.

In oncology, the knowledge of disease-specific molecular alterations is essential. The therapeutic value of a given target is proportional to the quantification of the role of this target in well-defined tumor types, when the druggability of a given target has been properly assessed and when its full clinical potential has been defined in terms of safety and efficacy.

An original NP with a novel mechanism of action has de facto more chances to be pushed into development than a NP directed at a conventional target. In the current era of targeted therapy, it is a challenge to develop an anticancer drug, even a structurally new NP, whose NP "in advance of its time", i.e. with a target unknown or poorly characterized at the pharmacological level, can be difficult to manipulate as well. A well defined molecular target is not always absolutely mandatory, but it is highly preferable [START_REF] Ganesan | The impact of natural products upon modern drug discovery[END_REF].

Conclusion

The impact of NP on anticancer drug discovery has been very high in the past century, it was certainly more limited for the past 8 years but will remain considerable for many more years, not only for cancer but also for diseases such as microbial and parasitic infections [START_REF] Butler | Mother Nature's gifts to diseases of man: the impact of natural products on anti-infective, anticholestemics and anticancer drug discovery[END_REF]. The approval of the aforementioned drugs ixabepilone, trabectedin and temsirolimus is emblematic of the evolution of the field of NP in oncology. The following points are important:

-The field is alive and well in academia but needs more supports in pharmaceutical companies and new NP-derived molecules in clinical trials.

-Conventional cytotoxic agents remain present and expected from clinicians, but with a preference for NP targeting pathways other than the tubulin/microtubule network or the DNA replication machinery, considered old-fashioned. There is an unquestionable demand for novel cytotoxic agents bringing not only gains in efficacy but decreasing the frequency of adverse events and offering a better convenience in administration [START_REF] Ismael | Novel cytotoxic drugs: old challenges, new solutions[END_REF]. In this sense, ixabepilone pursues the long established tradition of cytotoxic NP directed at microtubules. The fact that this compound originates from a microorganism, and not a plant as for the taxanes or vinca alkaloids, illustrates the increasing importance of microbial sources for the discovery of medically useful NP.

Plants remains a sure value but cultivated microorganisms will certainly play a dominant role in the future. Trabectedin is more than a classical DNA-alkylating agent; the downstream implication of the DNA repair machinery is critical for the drug action and possibly for patients selection.

-We have entered the era of targeted therapy with NP. This is probably the most salient feature of the field and a decisive step toward the demonstration that NP can continue to play a key role in modern antitumor pharmacology. The approval of temsirolimus, likely followed by one or two structurally related mTOR inhibitors in the near future, marks the transition between conventional chemotherapy with highly cytotoxic NP and more selective microbial products targeting a specific molecular pathway essential for the proliferation of cancer cells. Certainly in this particular case the chemotype is not new (everolimus has been known for years) but the first hurdle has been crossed to show that it makes sense to consider NP as a valuable source for innovative targeted therapeutic agents. During years 2000-2006, NP have been somewhat neglected and arguably underestimated by most pharmaceutical companies, to favor biologics and synthetic small molecules targeting a narrow ATP-pocket of protein kinases in particular. Nevertheless, during the same period the US approval success rate has remained low, around 8% for oncology [START_REF] Reichert | Development trends for new cancer therapeutics and vaccines[END_REF]

  5], remains one of the most important molecule of the anticancer armamentarium. The situation is comparable with mitomycin C, discovered in the mid-1950s[START_REF] Hata | Mitomycin, a new antibiotic from Streptomyces[END_REF], used clinically in the early 1960s and still prescribed nowadays for lung, gastric colorectal and pancreatic cancers for examples. This antibiotic, originally isolated from Streptomyces caespitosus, is a bioreductive drug that requires metabolic activation by cellular reductases, such as NAD(P)H:Quinone oxidoreductase-1 (NQO1), for activity. It is widely used systemically for the treatment of malignancies, and has gained popularity as topical adjunctive therapy in ocular and adnexal surgery over the past two decades. In the same vein, one can refer to bleomycin and actinomycin, two other old antitumor antibiotics still on the shelves of clinical oncologists. A parallel situation can be established with drugs derived from plants.

  resulting in arrested tumor cell division and apoptosis. But, in contrast to taxanes, ixabepilone has reduced susceptibility to resistance due to P-gp overexpression, tubulin mutations, and alterations in β-tubulin isotype expression. It demonstrated in vivo antitumor activity in a range of human tumor models, including tumors resistant to anthracyclines and taxanes[START_REF] Fornier | Ixabepilone, first in a new class of antineoplastic agents: the natural epothilones and their analogues[END_REF][START_REF] Lee | Preclinical discovery of ixabepilone, a highly active antineoplastic agent[END_REF][START_REF] Lee | Preclinical efficacy spectrum and pharmacokinetics of ixabepilone[END_REF]. In October 2007, ixabepilone administered by intravenous infusion, received FDA approval for the treatment of metastatic or locally advanced breast cancer. That is 12 years from the discovery of epothilone B to the launch of ixabepilone for the treatment of cancer. Other epothilone derivatives, such as sagopilone (ZK-EPO) and patupilone (epothilone B) are being developed[START_REF] Altmann | Epothilones as lead structures for new anticancer drugs-pharmacology, fermentation, and structure-activity-relationships[END_REF].-Trabectedin (Ecteinascidin-743, Yondelis ® developed by PharmaMar in partnership with Johnson & Johnson). ET-743 is a tetrahydroisoquinoline alkaloid derived from the Caribbean marine tunicate Ecteinascidia turbinata (sea-squirt) but now produced semi-synthetically starting from cyanosafracin B extracted after fermentation of the marine bacterium Pseudomonas fluorescens. It functions as a DNA minor groove alkylating agent specific of guanine residues at the N2 position, producing specific adducts that represent a unique challenge to the DNA repair machinery[START_REF] Soares | Replication and homologous recombination repair regulate DNA double-strand break formation by the antitumor alkylator ecteinascidin 743[END_REF]. Alkylation results in DNA bending which affects various transcription factors involved in cell proliferation, particularly via the transcription-coupled nucleotide excision repair system. Trabectedin has been recently

  A c c e p t e d M a n u s c r i p t 9 downstream effector of the oncogenic PI3K/Akt pathway and is a key regulator of translational initiation, offering thus a range of possible combinations with other targeted therapeutics, for the treatment of solid tumors and lymphoma. More than 100 clinical trials are ongoing to identify additional malignancies that respond to temsirolimus or other mTOR inhibitors[START_REF] Figlin | Mechanisms of disease: survival benefit of temsirolimus validates a role for mTOR in the management of advanced RCC[END_REF]. Along with the approved compound temsirolimus, there are two other mTOR inhibitors furthest along in development in oncology, everolimus and deferolimus. This class of rapamycin analogs has been known for many years. The immunosuppressant sirolimus (rapamycin, Rapamune ® ) is used in combination with other medications to prevent rejection of kidney transplants. Another derivative, tacrolimus, gained FDA approval for use in liver transplantation in 1994 and, approximately 3 years later, was approved for the prevention of acute rejection in kidney transplantation. Over the last decade tacrolimus has become the calcineurin inhibitor of choice for the prevention of rejection in renal transplantation.

A

  c c e p t e d M a n u s c r i p t 17 mechanism of action resembles that of classical molecules, in particular DNA-interacting drugs, topoisomerase inhibitors or tubulin/microtubule-interacting agents. But a brand new

-

  The development of marine NP is progressing actively. A marine microorganism has provided a new tumor-active drug (Trabectedin) now hemi-synthesized from a natural precursor (cyanosafracin B) extracted from a microorganism. The shift from macro to micro-organisms illustrates the evolution of the field.

A c c e p t e d M a n u s c r i p t 18 -

 18 The detailed understanding of the mechanism of action of a NP is essential.

  and the medical need is always high among cancer patients and their clinicians. The approval of the 3 new NP-derived drugs in 2007 hopefully announces a new departure for NP-based drug discovery in oncology. We are ready for this long awaited comeback.

Fig. 1 .

 1 Figures Legends

Fig. 2 .

 2 Fig. 2. The semi-synthetic vinca alkaloid vinorelbine, synthesized from the leaves of Catharanthus roseus, was first approved in 1989 in Europe for the i.v. treatment of non-small cell lung cancer. In 2001, the drug was approved as soft gelatin capsules for oral treatments of NSCLC.

Fig. 3 .

 3 Fig. 3. After a decade in the wilderness, in 2007 natural products make a comeback in oncology. Drugs recently approved for the treatment of cancers (year of first FDA approval)by category of origin. Note that, for the sake of clarity, all small molecules (e.g. aromatase inhibitors) are not mentioned.

Fig. 4 .

 4 Fig. 4. The multifunctionality of topoisomerase I and its pharmacological control. topoisomerase I carries three major biochemical functions: (i) control of DNA topology, via DNA cleavage and religation, an essential mechanism which can be altered by suppressors and poisons such as the clinically-used camptothecins, (ii) interaction with protein partners, including pro-apoptotic factors and (iii) regulation of RNA splicing via a kinase activity. The NP-derived antitumor drug NB-506 acts as a potent inhibitor of both kinase and relaxing activities of topoisomerase I, and modulates pre-mRNA splicing through inhibition of phosphorylation of the splicing factor SF2/ASF which is up-regulated in various human tumors. See text for more details.

  Figure 1

Figure 2 M

 2 Figure 2

Figure 2 A

 2 Figure 2

Acknowledgments

The author thanks many colleagues from the IRPF and PFO for fruitful discussions on natural products and reading of the manuscript.


Isabelle Buck

Franck Morceau

Christina Grigorakaki

Mario Dicato

Marc Diederich
email: marc.diederich@lbmcc.lu

Linking anemia to inflammation and cancer: the crucial role of TNFα α α α

Keywords: Epo, Erythropoietin, NF-κB, Nuclear Factor-kappa B, TNFα, Erythropoietin, erythroid differentiation, inflammation, cancer

Erythropoiesis is considered as a multistep and tightly regulated process under the control of a series of cytokines including erythropoietin (Epo). Epo activates specific signaling pathways and leads to activation of key transcription factors such as GATA-1, in order to ensure erythroid differentiation. Deregulation leads to a decreased number of red blood cells, a hemoglobin deficiency, thus a limited oxygen-carrying capacity in the blood. Anemia represents a frequent complication in various diseases such as cancer or inflammatory diseases. It reduces both quality of life and prognosis in patients. Tumor necrosis factor alpha (TNFα) was described to be involved in the pathogenesis of inflammation and cancer related anemia. Blood transfusions and erythroid stimulating agents (ESAs) including human recombinant Epo (rhuEpo) are currently used as efficient treatments. Moreover, the recently described conflicting effects of ESAs in distinct studies require further investigations on the molecular mechanisms involved in TNFα-caused anemia. The present study aims to evaluate the current knowledge and the importance of the effect of the proinflammatory cytokine TNFα on erythropoiesis in inflammatory and malignant conditions.

Introduction

The proinflammatory cytokine tumor necrosis factor (TNFα) was brought in connection with inflammation and cancer, two tightly linked research areas [START_REF] Balkwill | Smoldering and polarized inflammation in the initiation and promotion of malignant disease[END_REF][START_REF] Balkwill | Inflammation and cancer: back to Virchow?[END_REF][START_REF] Coussens | Inflammation and cancer[END_REF] and it was demonstrated that cancer-associated inflammation could promote tumor growth [START_REF] Balkwill | Smoldering and polarized inflammation in the initiation and promotion of malignant disease[END_REF][START_REF] Greten | IKKbeta links inflammation and tumorigenesis in a mouse model of colitis-associated cancer[END_REF][START_REF] Pikarsky | NF-kappaB functions as a tumour promoter in inflammation-associated cancer[END_REF]. TNFα expression has been confirmed in the tumor microenvironment of various malignancies [START_REF] Szlosarek | Tumour necrosis factor alpha: a potential target for the therapy of solid tumours[END_REF] and was categorized as a tumor promoter because of its effects on tumor initiation and progression [START_REF] Szlosarek | Tumour necrosis factor-alpha as a tumour promoter[END_REF][START_REF] Yan | Therapeutic potential of cytokine and chemokine antagonists in cancer therapy[END_REF]. Furthermore there are more and more drugs in clinical development that modulate TNFα function in a wide range of inflammatory diseases and cancers [START_REF] Yan | Therapeutic potential of cytokine and chemokine antagonists in cancer therapy[END_REF].

Cancer and inflammation related anemia were shown to be mediated by cytokine release, and particularly by TNFα, Interferon-β (IFN-β), and Interleukin-1 (IL-1) [START_REF] Bokemeyer | Anaemia in cancer patients: pathophysiology, incidence and treatment[END_REF].

Moreover, inhibition of colony-forming units-erythroid (CFU-E) in uremic patients with inflammatory disease due to TNFα and IFN-γ release has been reported [START_REF] Allen | Inhibition of CFU-E colony formation in uremic patients with inflammatory disease: role of IFN-gamma and TNF-alpha[END_REF].

Following anti-TNFα therapy, patients with rheumatoid arthritis showed improvement in anemic symptoms [START_REF] Papadaki | Anemia of chronic disease in rheumatoid arthritis is associated with increased apoptosis of bone marrow erythroid cells: improvement following anti-tumor necrosis factor-alpha antibody therapy[END_REF]. Since several years, human recombinant erythropoietin (rhuEpo) is a specific remedy administered against cancer-associated anemia. This treatment has a positive impact on hemoglobin levels and patient quality of life is improved. However, a preclinical background and some clinical data suggest a detrimental role of Epo in cancer by a possible stimulation of tumor growth.

Regulation of erythropoiesis

Hematopoiesis is the physiological process that leads to the formation of circulating blood cells from common hematopoietic stem cells (HSC) in the bone marrow. The different mature hematopoietic cells are usually classified in lymphoid and myeloid lineages. They are regulated by distinct cytokines acting on multipotential progenitors and their committed offspring [START_REF] Robb | Cytokine receptors and hematopoietic differentiation[END_REF][START_REF] Wickrema | Erythroid and megakaryocytic transformation[END_REF] (Fig. 1).

Erythropoiesis is a multistep event leading to the formation of erythrocytes. Erythroid differentiation arises from the myeloid root and is phenotypically characterized by the production of hemoglobin and expression of erythroid markers (Fig. 2). During differentiation from a multipotent common myeloid progenitor (CMP) to a bipotent megakaryocytic/erythroid progenitor (MEP), burst-forming units erythroid (BFU-E)

A c c e p t e d M a n u s c r i p t and CFU-E are the earliest identifiable erythroid progenitors in culture (Fig. 1). BFU-E and CFU-E are characterized by their in vitro ability to form colonies.

Erythropoiesis is a very dynamic and tightly regulated process by which 2*10 11 erythrocytes (lifespan of 100-120 days) are produced every day. Ferrous iron (Fe 2+ ) is essential for erythropoiesis as a major component of heme in hemoglobin as well as in the redox system of the respiratory chain. Hepcidin, a 25-amino acid peptide, is the main regulator of iron transport. A feedback loop involving the major cytokine for human erythropoiesis, Epo, regulates this physiological process, but other cytokines and nuclear hormones are also involved. IL-3 increases the number of BFU-E, whereas Stem Cell Factor (SCF) raises the number of cells within BFU-E and CFU-E (Fig. 1).

Kidney and liver are the main sites that produce the Epo in adult humans. The rate of Epo gene expression depends on the level of tissue oxygen through the availability of the hypoxia inducible factor (HIF). HIF heterodimer is composed of the oxygen sensitive HIF-1α and the constitutively expressed HIF-1β subunits. In hypoxic conditions HIF interacts with specific binding sites in the Epo enhancer. Oxygendependent prolyl hydroxylases control Epo variations in the kidney by regulating the stability of HIF-1α. The number of circulating erythrocytes is directly dependent on Epo amount in blood.

Epo is implicated in the control of cell survival, proliferation and differentiation within the erythroid pathway. It acts through its receptor (EpoR) in order to stimulate underlying cell signaling pathways including the Phosphatidylinositol 3 kinase (PI3K), the Janus kinase (JNK)/Signal-transducer and activator of transcription (STAT) and the mitogen-activated protein kinase (MAPK)/extracellular signal-related kinase pathways (ERK) [START_REF] Hodges | Pathophysiology of anemia and erythrocytosis[END_REF]. Moreover, Epo has been reported to modulate GATA-1 function in erythroid cells [START_REF] Zhao | Erythropoietin stimulates phosphorylation and activation of GATA-1 via the PI3-kinase/AKT signaling pathway[END_REF].

The survival of erythroid precursors and their terminal differentiation into red blood cells depends on Epo/EpoR interaction and GATA-1 transcription factor activity.

GATA-1 was first identified as a protein with binding capacity to the β-globin promoter [START_REF] Wall | The human beta-globin gene 3' enhancer contains multiple binding sites for an erythroid-specific protein[END_REF]. It is a member of the GATA family, which includes 6 members (GATA-1 to GATA-6). These transcription factors recognize the same DNA consensus sequence (A/T)GATA(A/G) and present two characteristic zinc finger motifs specific to the GATA family [START_REF] Merika | DNA-binding specificity of GATA family transcription factors[END_REF]. Three functional domains compose GATA-A c c e p t e d M a n u s c r i p t 1 protein: the N-terminal Zinc finger that supplies the stabilization and specificity of DNA binding and is responsible for the interactions with cofactors, the C-terminal Zinc finger that is essential for binding to the GATA consensus sequence of the DNA and the N-terminal activation domain [START_REF] Morceau | GATA-1: friends, brothers, and coworkers[END_REF]. GATA-1 is required for terminal erythroid maturation [START_REF] Ferreira | GATA1 function, a paradigm for transcription factors in hematopoiesis[END_REF]. Indeed, its crucial role in erythropoiesis was shown using GATA-1 null mouse embryos, which died between E10.5 and E12.5 from severe anemia due to a complete ablation of embryonic erythropoiesis [START_REF] Fujiwara | Arrested development of embryonic red cell precursors in mouse embryos lacking transcription factor GATA-1[END_REF]. Moreover, GATA-1-/-embryonic stem cells cannot contribute to definitive erythropoiesis [START_REF] Pevny | Erythroid differentiation in chimaeric mice blocked by a targeted mutation in the gene for transcription factor GATA-1[END_REF].

GATA-1 activity is dependent on protein-protein interactions, involving cofactors with either promoting or repressing activities [START_REF] Morceau | GATA-1: friends, brothers, and coworkers[END_REF] (Table 1). The transcription factor PU.1, an Ets family member of transcription factors, is required for the development of the myeloid and lymphoid lineages. Nevertheless, its inhibitory effect on GATA-1 activity can prevent erythroid differentiation. PU.1 and GATA-1 have a crossantagonistic relationship. Indeed, GATA-1 and PU.1 seem to functionally antagonize each other via direct physical interaction of their DNA-binding domains. PU.1 impairs GATA-1 by inhibiting its binding to DNA while GATA-1 inhibits PU.1 by preventing its interaction with c-Jun [START_REF] Rekhtman | Direct interaction of hematopoietic transcription factors PU.1 and GATA-1: functional antagonism in erythroid cells[END_REF].

In summary, GATA-1 activity is dependent on complex positive and negative interactions with transcriptional cofactors as well as posttranslational modifications leading to modulation of erythroid lineage-specific genes transcription. GATA-2, Nuclear Factor Erythroid-2 (NF-E2) and erythroid Krüppel-like factor (EKLF) are other specific transcription factors that have a major influence on erythroid differentiation and that are activated during erythropoiesis. GATA-2 is overexpressed during early hematopoiesis resulting in maintenance of the renewal capacity of erythroid progenitor cells. Its expression is then progressively repressed by the increasing expression of GATA-1 [START_REF] Grass | GATA-1dependent transcriptional repression of GATA-2 via disruption of positive autoregulation and domain-wide chromatin remodeling[END_REF]. NF-E2, is described to act as a major regulator of hemoglobin synthesis during erythropoiesis [START_REF] Andrews | Erythroid transcription factor NF-E2 is a haematopoietic-specific basicleucine zipper protein[END_REF], and EKLF, as a crucial factor in erythroid and megakaryocytic differentiation and maturation [START_REF] Lu | Retroviral integration within the Fli-2 locus results in inactivation of the erythroid transcription factor NF-E2 in Friend erythroleukemias: evidence that NF-E2 is essential for globin expression[END_REF][START_REF] Perkins | Fetal expression of a human Agamma globin transgene rescues globin chain imbalance but not hemolysis in EKLF null mouse embryos[END_REF] (Table 1).

Deregulation of

Tumor necrosis factor alpha

TNFα also known as cachectin or differentiation inducing factor (DIF), is a proinflammatory multifunctional cytokine, which is mainly produced by macrophages, but also by neutrophiles, fibroblasts, keratinocytes, astrocytes, Kupffer cells, smooth-muscle cells, T and B cells. It was initially described to induce hemorrhagic necrosis in transplanted tumors [START_REF] Carswell | An endotoxin-induced serum factor that causes necrosis of tumors[END_REF]. TNFα effects are principally mediated through two distinct receptors TNFα receptor (TNFR) I and II.

TNFα/TNFR interaction results in stimulation of the underlying cell signaling pathways that lead to nuclear factor kappa B (NF-κB), c-Jun N-terminal kinase (JNK), p38MAPK, or caspase activation. Thus TNFα simultaneously activates both apoptotic and anti-apoptotic or cell survival signals depending on the factors present in the receptor complex [START_REF] Aggarwal | Signalling pathways of the TNF superfamily: a double-edged sword[END_REF]. TNFα, which was discovered in B cells, is known as the most powerful activator of NF-κB transcription factor. NF-κB is known to bind its specific ten base pair consensus-binding site in order to regulate over 200 immune, growth, and inflammation genes.

TNFα, as well as other TNFα superfamily members, plays a role in hematopoiesis, host defense, immune surveillance, and proliferation. In this regard, TNFα deregulation leads to numerous diseases, including cancer [START_REF] Aggarwal | Signalling pathways of the TNF superfamily: a double-edged sword[END_REF][START_REF] Aggarwal | Inflammation and cancer: how hot is the link?[END_REF][START_REF] Pfeffer | Biological functions of tumor necrosis factor cytokines and their receptors[END_REF].

TNFα α α α and inflammation

A TNFα overproduction is involved in numerous chronic inflammatory diseases, such as rheumatoid arthritis [START_REF] Feldmann | Role of cytokines in rheumatoid arthritis[END_REF] chronic hepatitis C [START_REF] Knobler | TNF-{alpha}, chronic hepatitis C and diabetes: a novel triad[END_REF], or Crohn's disease [START_REF] Targan | A short-term study of chimeric monoclonal antibody cA2 to tumor necrosis factor alpha for Crohn's disease. Crohn's Disease cA2 Study Group[END_REF]. An increase in the TNFα level was described in diabetic patients to cause retinopathies [START_REF] Limb | Distribution of TNF alpha and its reactive vascular adhesion molecules in fibrovascular A c c e p t e d M a n u s c r i p t membranes of proliferative diabetic retinopathy[END_REF], while during pancreatitis, the release of TNFα leads to inflammation and cellular damage [START_REF] Friess | Growth factors and cytokines in pancreatic carcinogenesis[END_REF].

Currently with agent and disease [START_REF] Haraoui | Differentiating the efficacy of tumor necrosis factor inhibitors[END_REF][START_REF] Haraoui | The anti-tumor necrosis factor agents are a major advance in the treatment of rheumatoid arthritis[END_REF]. TNFα is thus leading to various biological phenomena implying different molecular mechanisms and is involved in different cellular responses. Although TNFα is considered to act as a proinflammatory cytokine, it was described as a positive and negative regulator of myeloid cell proliferation and differentiation [START_REF] Broxmeyer | The suppressive influences of human tumor necrosis factors on bone marrow hematopoietic progenitor cells from normal donors and patients with leukemia: synergism of tumor necrosis factor and interferon-gamma[END_REF][START_REF] Murase | Effect of recombinant human tumor necrosis factor on the colony growth of human leukemia progenitor cells and normal hematopoietic progenitor cells[END_REF][START_REF] Rusten | Tumor necrosis factor-alpha inhibits stem cell factor-induced proliferation of human bone marrow progenitor cells in vitro. Role of p55 and p75 tumor necrosis factor receptors[END_REF]. Effects of TNFα can be mediated either directly [START_REF] Rusten | Tumor necrosis factor (TNF)-alpha directly inhibits human erythropoiesis in vitro: role of p55 and p75 TNF receptors[END_REF] or indirectly by inducing other cells to produce cytokines, including hematopoietic growth factors [START_REF] Means | Inhibition of human colonyforming-unit erythroid by tumor necrosis factor requires accessory cells[END_REF][START_REF] Means | Inhibition of human erythroid colony-forming units by tumor necrosis factor requires beta interferon[END_REF].

Link between TNFα α α α, inflammation and cancer

Abnormal TNFα levels have been confirmed in tumor microenvironment [START_REF] Szlosarek | Tumour necrosis factor alpha: a potential target for the therapy of solid tumours[END_REF].

Moreover, this cytokine is paradoxically able to induce necrosis and to promote tumor development, depending on the levels of TNFα in distinct settings [START_REF] Balkwill | Tumor necrosis factor or tumor promoting factor?[END_REF]. When TNFα is secreted by tumors and tumor-associated macrophages, it promotes tumor growth and stimulates angiogenesis, whereas when it is administered therapeutically at high doses, it induces an increased permeability of tumor vasculature. Thus, recombinant TNFα, as a tumor regressing agent, is approved in Europe to be administered locoregionally at supraphysiological levels as a therapy for soft tissue sarcoma [START_REF] Van Etten | Fifty tumor necrosis factor-based isolated limb perfusions for limb salvage in patients older than 75 years with limb-threatening soft tissue sarcomas and other extremity tumors[END_REF].

Using murine models, it was shown that inflammation-associated hepatocellular carcinogenesis involved the activation of the tumor promoter NF-κB via the production of TNFα [START_REF] Pikarsky | NF-kappaB functions as a tumour promoter in inflammation-associated cancer[END_REF]. Moreover several reports associate detection of abnormally high levels of TNFα protein and/or constitutively active NF-κB in cancer patients with a wide range of tumor types [START_REF] Mantovani | Serum levels of leptin and proinflammatory cytokines in patients with advanced-stage cancer at different sites[END_REF], including kidney [START_REF] Yoshida | Interleukin-6, tumour necrosis factor alpha and interleukin-1beta in patients with renal cell carcinoma[END_REF], breast [START_REF] Leek | Association of tumour necrosis factor alpha and its receptors with thymidine phosphorylase expression in invasive breast carcinoma[END_REF], asbestosis induced lung [START_REF] Partanen | Tumour necrosis factor-alpha (TNFalpha) in patients who have asbestosis and develop cancer[END_REF], and prostate cancers [START_REF] Pfitzenmaier | Elevation of cytokine levels in cachectic patients with prostate carcinoma[END_REF]. Suppression of constitutively active NF-κB results in cell proliferation arrest and apoptosis, indicating a crucial role for NF-κB in proliferation and survival [START_REF] Aggarwal | Nuclear factor-kappaB: the enemy within[END_REF]. Furthermore chronic bioavailability of TNFα has been correlated with enhanced invasive activities as well as survival of neoplastic cells [START_REF] Balkwill | Tumor necrosis factor or tumor promoting factor?[END_REF]. Within groups of patients with the same tumor type, higher levels of TNFα have been correlated with advanced tumor stage, greater complications, and shorter survival time [START_REF] Bossola | Serum tumour necrosis factor-alpha levels in cancer patients are discontinuous and correlate with weight loss[END_REF]. Moreover various cytokines, including TNFα, are overexpressed in pancreatic cancer cells, leading to an NF-κB activation and as a consequence, to A c c e p t e d M a n u s c r i p t cell growth by inhibiting apoptosis [START_REF] Chang | Nuclear factor kappaB protects pancreatic beta-cells from tumor necrosis factor-alphamediated apoptosis[END_REF]. TNFα also appears as a growth factor regulated by NF-κB in Hodgkin's lymphoma, T cell lymphoma and glioma [START_REF] Pahl | Activators and target genes of Rel/NF-kappaB transcription factors[END_REF].

As Rudolf Virchow already suspected in 1863, inflammation and cancer have to be handled together [START_REF] Balkwill | Smoldering and polarized inflammation in the initiation and promotion of malignant disease[END_REF][START_REF] Coussens | Inflammation and cancer[END_REF]. Cancer-associated inflammation includes the expression of cytokines such as TNFα or IL-1 by tumor-associated macrophages, stimulating tumor growth [START_REF] Allavena | The inflammatory micro-environment in tumor progression: the role of tumor-associated macrophages[END_REF]. As TNFα was categorized as a tumor promoter, it is not astonishing that cytokine antagonists as well as NF-κB inhibitors are already used in cancer therapy and prevention [START_REF] Szlosarek | Tumour necrosis factor-alpha as a tumour promoter[END_REF][START_REF] Yan | Therapeutic potential of cytokine and chemokine antagonists in cancer therapy[END_REF][START_REF] Balkwill | Tumor necrosis factor or tumor promoting factor?[END_REF].

Deregulation of erythropoiesis by TNFα α α α in inflammation and cancer.

Besides the proinflammatory, proliferative and apoptotic properties, TNFα was also described as an inhibitor of the erythroid differentiation in vitro and in vivo [START_REF] Broxmeyer | The suppressive influences of human tumor necrosis factors on bone marrow hematopoietic progenitor cells from normal donors and patients with leukemia: synergism of tumor necrosis factor and interferon-gamma[END_REF][START_REF] Murase | Effect of recombinant human tumor necrosis factor on the colony growth of human leukemia progenitor cells and normal hematopoietic progenitor cells[END_REF][START_REF] Rusten | Tumor necrosis factor (TNF)-alpha directly inhibits human erythropoiesis in vitro: role of p55 and p75 TNF receptors[END_REF][START_REF] Roodman | Mechanisms of erythroid suppression in the anemia of chronic disease[END_REF] (Fig. 3). Its expression is associated with the hematologic diseases Fanconi anemia (FA) [START_REF] Dufour | TNFalpha and IFN-gamma are overexpressed in the bone marrow of Fanconi anemia patients and TNF-alpha suppresses erythropoiesis in vitro[END_REF], myelodysplastic syndromes [START_REF] Gersuk | A role for tumour necrosis factor-alpha, Fas and Fas-Ligand in marrow failure associated with myelodysplastic syndrome[END_REF], aplastic anemia [START_REF] Dufour | Interferon gamma and tumour necrosis factor alpha are overexpressed in bone marrow T lymphocytes from paediatric patients with aplastic anaemia[END_REF] and anemia due to chronic diseases [START_REF] Imagawa | A GATA-specific inhibitor (K-7174) rescues anemia induced by IL-1beta, TNFalpha, or L-NMMA[END_REF]. Indeed, in FA patients, TNFα is significantly overexpressed in stimulated marrow mononuclear cells, which leads to a suppression of erythropoiesis. In bone marrow cultures, the addition of anti-TNFα increases the size and the number of CFU-E and BFU-E grown from FA patients but not from healthy controls. This indicates that FA subjects have a marrow TNFα activity that inhibits erythropoiesis in vitro. TNFα plays a relevant role in the pathogenesis of erythroid failure in FA patients [START_REF] Dufour | TNFalpha and IFN-gamma are overexpressed in the bone marrow of Fanconi anemia patients and TNF-alpha suppresses erythropoiesis in vitro[END_REF]. Several in vitro studies revealed the inhibitory effects of this cytokine on hematopoietic progenitor cell growth [START_REF] Broxmeyer | The suppressive influences of human tumor necrosis factors on bone marrow hematopoietic progenitor cells from normal donors and patients with leukemia: synergism of tumor necrosis factor and interferon-gamma[END_REF][START_REF] Murase | Effect of recombinant human tumor necrosis factor on the colony growth of human leukemia progenitor cells and normal hematopoietic progenitor cells[END_REF][START_REF] Rusten | Tumor necrosis factor (TNF)-alpha directly inhibits human erythropoiesis in vitro: role of p55 and p75 TNF receptors[END_REF][START_REF] Roodman | Mechanisms of erythroid suppression in the anemia of chronic disease[END_REF][START_REF] Dufour | TNFalpha and IFN-gamma are overexpressed in the bone marrow of Fanconi anemia patients and TNF-alpha suppresses erythropoiesis in vitro[END_REF][START_REF] Xiao | Tumor necrosis factor-alpha inhibits generation of glycophorin A+ cells by CD34+ cells[END_REF]. It was shown that the inhibition of human CFU-E by TNFα requires IFN-β, which is produced by macrophages in response to TNFα. TNFα was also shown to have a direct inhibitory effect on Epo-induced generation of GPA positive cells from CD34+ progenitors, leading to the suppression of erythropoiesis and the reduction of the proliferation capacity of GPA positive cells [START_REF] Xiao | Tumor necrosis factor-alpha inhibits generation of glycophorin A+ cells by CD34+ cells[END_REF]. Interestingly, TNFα is also believed to play a critical role in many forms of cancer [START_REF] Bokemeyer | Anaemia in cancer patients: pathophysiology, incidence and treatment[END_REF][START_REF] Kurzrock | The role of cytokines in cancer-related fatigue[END_REF] and inflammation related anemia [START_REF] Papadaki | Anemia of chronic disease in rheumatoid arthritis is associated with increased apoptosis of bone marrow erythroid cells: improvement following anti-tumor necrosis factor-alpha antibody therapy[END_REF]. Indeed anemia is considered as a common symptom induced by inflammation and cancer pathologies. In patients with B-cell chronic lymphocytic leukemia suffering from anemia, the serum levels of TNFα were significantly higher than in those without anemia [START_REF] Capalbo | Evaluation of tumor necrosis factor-alpha and erythropoietin serum levels in B-cell chronic lymphocytic leukemia patients with anemia[END_REF]. The incidence of anemia was shown to vary with A c c e p t e d M a n u s c r i p t tumor type, stage and patient age. Up to one third of patients had anemia at diagnosis [START_REF] Knight | Prevalence and outcomes of anemia in cancer: a systematic review of the literature[END_REF]. This number increases after chemotherapy [START_REF] Tas | Anemia in oncology practice: relation to diseases and their therapies[END_REF]. Cancer-associated anemia was shown to reduce survival of patients regardless of tumor type [START_REF] Caro | Anemia as an independent prognostic factor for survival in patients with cancer: a systemic, quantitative review[END_REF]. Moreover, the quality of life is considerably affected and is associated with a range of symptoms including fatigue, depression, and dizziness [START_REF] Pronzato | Cancer-related anaemia management in the 21st century[END_REF]; thus pro-inflammatory cytokines were recently suggested as the common denominator for cancer related fatigue [START_REF] Engert | Recombinant human erythropoietin in oncology: current status and further developments[END_REF].

Inflammation associated anemia is considered as a main symptom of patients with inflammatory disorders [START_REF] Andrews | Anemia of inflammation: the cytokine-hepcidin link[END_REF]. Prior to the use of erythroid stimulating agents (ESAs), the most frequent treatment of cancer related anemia was blood transfusion. Clinical trials established erythropoietin's ability to increase hemoglobin levels and reduce transfusion requirements [START_REF] Balkwill | Inflammation and cancer: back to Virchow?[END_REF][START_REF] Coussens | Inflammation and cancer[END_REF]. However, the conflicting effects of ESAs were recently described in distinct studies [START_REF] Blau | Erythropoietin in cancer: presumption of innocence?[END_REF][START_REF] Jelkmann | Developments in the therapeutic use of erythropoiesis stimulating agents[END_REF][START_REF] Rizzo | Use of epoetin and darbepoetin in patients with cancer: 2007 American Society of Hematology/American Society of Clinical Oncology clinical practice guideline update[END_REF]. Indeed, besides cardiovascular and thromboembolic events in erythropoietin-treated patients, several phase II and III trials showed a significant deterioration of cancer patients survival [START_REF] Henke | Erythropoietin to treat head and neck cancer patients with anaemia undergoing radiotherapy: randomised, double-blind, placebo-controlled trial[END_REF][START_REF] Leyland-Jones | Maintaining normal hemoglobin levels with epoetin alfa in mainly nonanemic patients with metastatic breast cancer receiving first-line chemotherapy: a survival study[END_REF][START_REF] Wright | Randomized, double-blind, placebo-controlled trial of erythropoietin in nonsmall-cell lung cancer with disease-related anemia[END_REF].

Unexpectedly, the increased mortality came from accelerated progression of cancer.

This reproducible effect was attributed to erythropoietin. Thus, the use of erythropoietin in cancer patients might increase the risk of cancer-associated death. It is suggested that additional phase III trials should be performed to determine whether erythropoietin is safe when used in accordance with FDA-approved indications. One claims that waiting for these further studies to modify or stop Epo treatments could have detrimental impact on many cancer patients [START_REF] Blau | Erythropoietin in cancer: presumption of innocence?[END_REF]. In this context, controversial effects of erythropoietin in cancer-related anemia makes necessary to further investigate the molecular mechanisms involved in anemia and to identify new targets for drug development as well as to detect more significant predictors. In order to improve quality of life, several drugs are under investigation for the treatment of different forms of anemia. Jelkmann reviewed several anti-anemic drugs and techniques based on Epo gene expression [START_REF] Jelkmann | Developments in the therapeutic use of erythropoiesis stimulating agents[END_REF][START_REF] Jelkmann | Control of erythropoietin gene expression and its use in medicine[END_REF].

Additionally, our group previously reported that TNFα-mediated inhibition of K562 cell differentiation was correlated to GATA-1 downregulation [START_REF] Morceau | Tumor necrosis factor alpha inhibits aclacinomycin A-induced erythroid differentiation of K562 cells via GATA-1[END_REF], GATA-1/GATA-2 unbalance in favor of GATA-2 as well as a decrease in the acetylation status of GATA-1 [START_REF] Buck | Tumor necrosis factor alpha inhibits erythroid differentiation in human erythropoietin-dependent cells involving p38 MAPK pathway, GATA-1 and FOG-1 downregulation and GATA-2 upregulation[END_REF][START_REF] Buck | The inhibitory effect of the proinflammatory cytokine TNFalpha on erythroid differentiation involves erythroid transcription factor modulation[END_REF]. Moreover we suggested a role for p38 in the inhibition of erythroid differentiation by TNFα, in correlation with a reversal of important erythroid transcription factors [START_REF] Buck | Tumor necrosis factor alpha inhibits erythroid differentiation in human erythropoietin-dependent cells involving p38 MAPK pathway, GATA-1 and FOG-1 downregulation and GATA-2 upregulation[END_REF]. Miwatashi and colleagues already used a novel p38

Conclusions

Erythropoiesis is a tightly regulated, complex physiological process leading to the formation of erythrocytes from a pluripotent hematopoietic stem cell. Deregulation can lead to various complications, including anemia. Anemia represents a frequent complication in cancer patients, as well as in patients suffering from inflammatory diseases. Proinflammatory cytokines seem to be overexpressed in these diseases.

Anemia considerably affects quality of life and is even considered as an independent bad prognostic factor. On the other hand some studies showed that the use of recombinant Epo as a treatment for cancer related anemia could be inappropriate for cancer patients. For these reasons the molecular mechanisms behind the inhibitory effect of TNFα on erythroid differentiation need to be further elucidated in order to find potential new, and more pointed therapeutic targets for inflammation and cancer related anemia. In this respect, investigations using hematopoietic stem cell culture systems should allow to better understand the impact of TNFα on the control of erythropoiesis by identifying which specific cellular process is affected, including differentiation and/or apoptosis regulation.

  Epo or other key factors of erythroid differentiation can lead to major changes in red blood cell number, and subsequent decrease in the oxygen-carrying capacity of the blood. Erythrocytosis are disorders resulting in an excessively high

  A c c e p t e d M a n u s c r i p t level of erythrocytes, whereas anemia is characterized by a qualitative or quantitative deficiency of hemoglobin. Anemia is clinically defined by a hemoglobin (Hb) level inferior to 12g/dL.

  , three marketed TNFα antagonists [etanercept (Enbrel®), infliximab (Remicade®), and adalimumab (Humira®)] are indicated in diseases characterized by A c c e p t e d M a n u s c r i p t abnormally elevated TNFα levels. Moreover the effectiveness of the treatments varies

A c c e

  p t e d M a n u s c r i p t inhibitor, N-[4-[2-Ethyl-4-(3-methylphenyl)-1,3-thiazol-5-yl]-2-pyridyl] benzamide(TAK-715), as an anti-TNFα drug for the treatment of rheumatoid arthritis, presenting anemic complications[START_REF] Papadaki | Anemia of chronic disease in rheumatoid arthritis is associated with increased apoptosis of bone marrow erythroid cells: improvement following anti-tumor necrosis factor-alpha antibody therapy[END_REF][START_REF] Miwatashi | Novel inhibitor of p38 MAP kinase as an anti-TNF-alpha drug: discovery of N-[4-[2-ethyl-4-(3-methylphenyl)-1,3-thiazol-5-yl]-2-pyridyl]benzamide (TAK-715) as a potent and orally active anti-rheumatoid arthritis agent[END_REF].

Figure 1 :

 1 Figure 1: Hematopoiesis and the role of cytokines. Cytokines act both on multipotential progenitors and their committed offspring. CLP, common lymphoid progenitor; CMP, common myeloid progenitor; HSC, hematopoietic stem cell; GMP, granulocyte-macrophage progenitor; MEP, megakaryocyte erythroid progenitor;BFU-E, burst-forming units-erythroid; CFU-E, colony-forming units-erythroid; CFU-Meg, colony-forming units-megakaryocyte; MPP, multipotent progenitor; IL, Interleukin; SCF, stem cell factor; GM-CSF, granulocyte-macrophage colony stimulating factor; G-CSF, granulocyte colony stimulating factor; M-CSF, macrophage colony stimulating factor; TPO, thrombopoietin; Epo, Erythropoietin (Adapted from[START_REF] Robb | Cytokine receptors and hematopoietic differentiation[END_REF][START_REF] Wickrema | Erythroid and megakaryocytic transformation[END_REF] with modifications).

Figure 2 :

 2 Figure 2: Stages of mammalian erythropoiesis and corresponding expression of erythroid specific markers. The relative sizes and morphologic appearances of erythroid cells at various stages of differentiation: common myeloid progenitor (CMP), megakaryocyte erythroid progenitor (MEP), burst-forming units-erythroid (BFU-E), colony-forming units-erythroid (CFU-E), proerythroblasts (Pro EB), basophilic erythroblasts (Baso EB), polychromatophilic erythroblasts (Poly EB),orthochromatic erythroblasts (Ortho EB), reticulocytes (RET), and erythrocytes (Eryt). Erythroid markers are represented in red and their periods of expression with gray lines: erythropoietin receptor (EpoR), Glycophorin A (GPA), Transferrin receptors (TFRC), δ-aminolevulinate synthase (δ-ALA-S) (Adapted from[START_REF] Koury | New insights into erythropoiesis[END_REF]).

Figure 3 :

 3 Figure 3: Pathophysiology of anemia. Tumor cells act on erythrocytes through macrophages by cytokine release, which leads to impaired erythropoiesis. Released cytokines can affect BFU-E and CFU-E proliferation, iron utilization and Epoproduction. TNFα can also affect erythrocyte half-life. TNFα, tumor necrosis factor alpha; IFN, Interferon; IL, Interleukin; BFU-E, burst-forming units-erythroid; CFU-E, colony-forming units-erythroid; Epo, Erythropoietin (Adapted from[START_REF] Engert | Recombinant human erythropoietin in oncology: current status and further developments[END_REF]).

Figure A c c e p t e d M a n u s c r i p

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Acknowledgements

Research in MD's lab is supported by the 'Recherche Cancer et Sang' foundation, the 'Recherches Scientifiques Luxembourg' association, Télévie and Action Lions Vaincre le Cancer. I.B. is supported by 'Action Lions Vaincre le Cancer' and C.G. by an AFR grant from FNR Luxembourg. The authors thank M. Groos for collaboration and 'Een Häerz fir kriibskrank Kanner' association for continuous support.

Factor name Effect References

GATA-1 + [START_REF] Fujiwara | Arrested development of embryonic red cell precursors in mouse embryos lacking transcription factor GATA-1[END_REF] GATA-2 +/- [START_REF] Tsai | Transcription factor GATA-2 is required for proliferation/survival of early hematopoietic cells and mast cell formation, but not for erythroid and myeloid terminal differentiation[END_REF] NF-E2 (Nuclear Factor Erythroid 2) + [START_REF] Shivdasani | Erythropoiesis and globin gene expression in mice lacking the transcription factor NF-E2[END_REF] FOG (Friend of GATA) -1 + [START_REF] Tsang | FOG, a multitype zinc finger protein, acts as a cofactor for transcription factor GATA-1 in erythroid and megakaryocytic differentiation[END_REF] Lmo2 (LIM-only protein 2) (Rbtn2) + [START_REF] Wadman | The LIM-only protein Lmo2 is a bridging molecule assembling an erythroid, DNA-binding complex which includes the TAL1, E47, GATA-1 and Ldb1/NLI proteins[END_REF] p300/CBP + [START_REF] Blobel | CREB-binding protein cooperates with transcription factor GATA-1 and is required for erythroid differentiation[END_REF] EKLF (Erythroid Kruppel like factor) + [START_REF] Gregory | Functional interaction of GATA1 with erythroid Kruppellike factor and Sp1 at defined erythroid promoters[END_REF] PU.1 (SPI1) - [START_REF] Rekhtman | Direct interaction of hematopoietic transcription factors PU.1 and GATA-1: functional antagonism in erythroid cells[END_REF] c-myb . [START_REF] Matsumura | Biologic significance of GATA-1 activities in Ras-mediated megakaryocytic differentiation of hematopoietic cell lines[END_REF] Table