L'identification des cellules souches cancéreuses (CSC) cette dernière décennie a permis l'émergence de nouveaux espoirs. Néanmoins, leur caractérisation a mis en évidence une certaine résistance aux traitements anticancéreux tels que la radiothérapie. De plus, il a récemment été montré que certaines cellules non-CSC pouvaient réacquérir le phénotype de cellules souches cancéreuses sous l'effet de traitement anticancéreux, enrichissant d'autant la population résistante. Nous proposons dans cette revue de faire un tour d'horizon des différentes propriétés des CSC et tout particulièrement des mécanismes de radiorésistance, de reprogrammation et des approches thérapeutiques envisagées pour outrepasser ces résistances.

There is accumulating evidence that TrkA and its ligand Nerve Growth Factor (NGF) are involved in cancer development. Staurosporine derivatives such as K252a and lestaurtinib have been developed to block TrkA kinase signaling, but no clinical trial has fully demonstrated their therapeutic efficacy. Therapeutic failures are likely due to the existence of intrinsic signaling pathways in cancer cells that impede or bypass the effects of TrkA tyrosine kinase inhibitors. To verify this hypothesis, we combined different approaches including mass spectrometry proteomics, co-immunoprecipitation and proximity ligation assays. We found that NGF treatment induced CD44 binding to TrkA at the plasma membrane and subsequent activation of the p115RhoGEF/RhoA/ROCK1 pathway to stimulate breast cancer cell invasion. The NGF-induced CD44 signaling was independent of TrkA kinase activity. Moreover, both TrkA tyrosine kinase inhibition with lestaurtinib and CD44 silencing with siRNA inhibited cell growth in vitro as well as tumor development in mouse xenograft model; combined treatment significantly enhanced the antineoplastic effects of either treatment alone. Altogether, our results demonstrate that NGF-induced tyrosine kinase independent TrkA signaling through CD44 was sufficient to maintain tumor aggressiveness. Our findings provide an alternative mechanism of cancer resistance to lestaurtinib and indicate that dual inhibition of CD44 and TrkA tyrosine kinase activity may represent a novel therapeutic strategy.

L'identification des cellules souches cancereuses (CSC) dans les tumeurs hematologiques et solides a conduit a de nombreuses etudes fondamentales et translationnelles. Les cellules cancereuses presentent cependant une certaine plasticite. En effet, les cellules cancereuses non souches (non-CSC) differenciees peuvent generer des CSC en reponse a divers stimuli. Ainsi, la radiotherapie (RT) entraine l'induction de CSC a partir de non-CSC, in vitro. Cette reprogrammation pourrait participer aux resistances aux traitements et aux recidives. Malgre tout, les mecanismes a l'origine de la reprogrammation restent meconnus, et il parait essentiel d'identifier des cibles pour prevenir l'apparition de CSC. Pendant ma these, j'ai montre que le milieu de non-CSC irradiees induit la reprogrammation en CSC mammaires. J'ai pu voir que la RT entraine la secretion specifique de chimiokines, dont CXCL1 et CCL5. Leur inhibition et le traitement par proteine recombinante ont permis de montrer l'implication de CXCL1, CCL5 et leurs recepteurs dans la reprogrammation in vitro. De plus, l'inhibition in vivo de CXCL1 et CCL5, combinee a la RT dans un modele murin de xenogreffes induit une augmentation de la survie. Enfin, l'analyse transcriptomique des bases de donnees cliniques a demontre une correlation entre l’expression des chimiokines et leurs recepteurs avec des signatures de CSC et de sous-types plus agressifs dans le cancer du sein, et une survie sans metastases diminuee. Mes resultats indiquent un role des chimiokines dans la reprogrammation de non-CSC en CSC et qu'elles peuvent constituer de nouvelles cibles therapeutiques, en combinaison avec les traitements traditionnels.

The Trk family of neurotrophin tyrosine kinase receptors is emerging as an important player in carcinogenic progression in non-neuronal tissues. Here, we show that breast tumors present high levels of TrkA and phospho-TrkA compared to normal breast tissues. To further evaluate the precise functions of TrkA overexpression in breast cancer development, we have performed a series of biological tests using breast cancer cells that stably overexpress TrkA. We show that (1) TrkA overexpression promoted cell growth, migration and invasion in vitro; (2) overexpression of TrkA per se conferred constitutive activation of its tyrosine kinase activity; (3) signal pathways including PI3K-Akt and ERK/p38 MAP kinases were activated by TrkA overexpression and were required for the maintenance of a more aggressive cellular phenotype; and (4) TrkA overexpression enhanced tumor growth, angiogenesis and metastasis of xenografted breast cancer cells in immunodeficient mice. Moreover, recovered metastatic cells from the lungs exhibited enhanced anoikis resistance that was abolished by the pharmacological inhibitor K252a, suggesting that TrkA-promoted breast tumor metastasis could be mediated at least in part by enhancing anoikis resistance. Together, these results provide the first direct evidence that TrkA overexpression enhances the tumorigenic properties of breast cancer cells and point to TrkA as a potential target in breast cancer therapy.

Tamoxifen (TAM), is widely used as a single agent in adjuvant treatment of breast cancer. Here, we investigated the effects of TAM in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in estrogen receptor-α (ER-α)-positive and -negative breast cancer cells. We showed that cotreatment with TAM and TRAIL synergistically induced apoptosis regardless of ER-α status. By contrast, cotreatment did not affect the viability of normal breast epithelial cells. Cotreatment with TAM and TRAIL in breast cancer cells decreased the levels of antiapoptotic proteins including FLIPs and Bcl-2, and enhanced the levels of proapoptotic proteins such as FADD, caspase 8, tBid, Bax and caspase 9. Furthermore, cotreatment-induced apoptosis was efficiently reduced by FADD- or Bid-siRNA, indicating the implication of both extrinsic and intrinsic pathways in synergistic apoptosis induction. Importantly, cotreatment totally arrested tumor growth in an ER-α-negative MDA-MB-231 tumor xenograft model. The abrogation of tumor growth correlated with enhanced apoptosis in tumor tissues. Our findings raise the possibility to use TAM in combination with TRAIL for breast cancers, regardless of ER-α status.

Sialyl-Tn is a carbohydrate antigen overexpressed in several epithelial cancers, including breast cancer, and usually associated with poor prognosis. Sialyl-Tn is synthesized by a CMP-Neu5Ac: GalNAc alpha 2,6-sialyltransferase: CMP-Neu5Ac: R-GalNAc alpha 1-O-Ser/Thr alpha 2,6-sialyltransferase(EC2.4.99.3) (ST6GalNAc I), which transfers a sialic acid residue in alpha 2,6-linkage to the GalNAc alpha 1-O-Ser/Thr structure. However, established breast cancer cell lines express neither ST6GalNAc I nor sialyl-Tn. We have previously shown that stable transfection of MDA-MB-231, a human breast cancer cell line, with ST6GalNAc I cDNA induces sialyl-Tn antigen (STn) expression. We report here the modifications of the O-glycosylation pattern of a MUC1-related recombinant protein secreted by MDA-MB-231 sialyl-Tn positive cells. We also show that sialyl-Tn expression and concomitant changes in the overall O-glycan profiles induce a decrease of adhesion and an increase of migration of MDA-MB-231. Moreover, STn positive clones exhibit an increased tumour growth in severe combined immunodeficiency ( SCID) mice. These observations suggest that modification of the O-glycosylation pattern induced by ST6GalNAc I expression are sufficient to enhance the tumourigenicity of MDA-MB-231 breast cancer cells.

Inhibitors of histone deacetylase (HDAC) are considered as potential anticancer agents. We have previously demonstrated that an inhibitor of HDAC, sodium butyrate (NaB), induces apoptosis of breast cancer cells in a P53-independent and P21(waf1)-dependent manner. In this study, we showed that tumor necrosis factor-alpha (TNF-alpha), TNF-related apoptosis-inducing ligand (TRAIL), and anti-Fas agonist antibody potentiated NaB-induced growth inhibition through synergistic induction of apoptosis in breast cancer cell lines (MCF-7, T47-D, and BT-20). In MCF-7 cells, NaB increased the expression of death receptors; NaB alone or in combination with TNF-alpha, TRAIL, and anti-Fas agonist antibody increased the levels of Bid, tBid, and that of cytosolic cytochrome c. Synergistic induction of apoptosis was strongly inhibited by dominant-negative Fas-associated death domain (FADD) and inhibitors of caspases-8 and -9, indicating that potentiation of apoptosis involved key elements of death receptors' signaling pathways. Moreover, cotreatment of NaB and ligands of death receptors up-regulated the levels of P21(waf1) and that of proliferating cell nuclear antigen (PCNA) associated with P21(waf1). Transient transfections of p21(waf1) antisense or p21(waf1) deficient for its interaction with PCNA abolished synergistic induction of apoptosis. This suggested that potentiation of apoptosis by cotreatments required P21(waf1) and its interaction with PCNA. Since breast tumors contain rarely p21 mutations, our results may open interesting prospects in the fight against breast cancer.

This study was performed to determine the effect and action mechanisms of sodium butyrate (NaB) on the growth of breast cancer cells. Butyrate inhibited the growth of all breast cancer cell lines analysed. It induced cell cycle arrest in G1 and apoptosis in MCF‐7, MCF‐7ras, T47‐D, and BT‐20 cells, as well as arrest in G2/M in MDA‐MB‐231 cells. Transient transfection of MCF‐7 and T47‐D cells with wild‐type and antisense p53 did not modify butyrate‐induced apoptosis. Pifithrin‐α, which inhibits the transcriptional activity of P53, did not modify cell growth or apoptosis of MCF‐7 and T47‐D cells treated with butyrate. These results indicate that P53 was not involved in butyrate‐induced growth inhibition of breast cancer cells. Treatment of MCF‐7 cells with anti‐Fas agonist antibody induced cell death, indicating that Fas was functional in these cells. Moreover, butyrate potentiated Fas‐induced apoptosis, as massive apoptosis was observed rapidly when MCF‐7 cells were treated with butyrate and anti‐Fas agonist antibody. In addition, butyrate‐induced apoptosis in MCF‐7 cells was considerably reduced by anti‐Fas antagonist antibody. Western blot analysis showed that butyrate increased Fas and Fas ligand levels (Fas L), indicating that butyrate‐induced apoptosis may be mediated by Fas signalling. These results demonstrate that butyrate inhibited the growth of breast cancer cells in a P53‐independent manner. Moreover, it induced apoptosis via the Fas/Fas L system and potentiated Fas‐triggered apoptosis in MCF‐7 cells. These findings may open interesting perspectives in human breast cancer treatment strategy. British Journal of Pharmacology (2002) 135, 79–86; doi:10.1038/sj.bjp.0704456

Fas/Fas ligand (Fas L) death pathway is an important mediator of apoptosis. Deregulation of Fas pathway is reported to be involved in the immune escape of breast cancer and the resistance to anti-cancer drugs. In this study, we demonstrated that conditioned medium by normal breast epithelial cells (NBEC-CM) induced apoptosis of MCF-7 and T-47D Fas-sensitive cells but had no effect on MDA-MB-231 Fas-resistant cells. Inhibition of PI3 kinase or NF-κB by specific inhibitors or transient transfections restored the sensitivity of MDA-MB-231 cells to NBEC-induced apoptosis. Moreover, the constitutive activation of NF-κB was controlled by PI3 kinase because inhibition of PI3 kinase reduced NF-κB activity. Inducible activation of NF-κB rendered MCF-7 cells resistant to NBEC-CM- and Fas agonist antibody-triggered apoptosis. Therefore, constitutive or inducible activation of PI3 kinase and/or NF-κB in breast cancer cells rendered them resistant to NBEC-triggered apoptosis. In addition, Fas neutralizing antibody and dominant negative Fas abolished NBEC-triggered apoptosis. Western blot and confocal microscopy analysis showed an increase of membrane Fas/Fas L when cells were induced into apoptotis by NBEC-CM. Taken together, these data show that NBEC induced apoptosis in breast cancer cells via Fas signaling.

Sialyl-Tn antigen (STn) is a cancer associated carbohydrate antigen over-expressed in several cancers including breast cancer, and currently associated with more aggressive diseases and poor prognosis. However, the commonly used breast cancer cell lines (MDA-MB-231, T47-D and MCF7) do not express STn antigen. The key step in the biosynthesis of STn is the transfer of a sialic acid residue in alpha2,6-linkage to GalNAcalpha-O-Ser/Thr. This reaction is mainly catalyzed by a CMP-Neu5Ac GalNAc alpha2,6-sialyltransferase: ST6GalNAc 1. In order to generate STn-positive breast cancer cells, we have cloned a cDNA encoding the full-lenght human ST6GaINAc I from HT-29-MTX cells. The stable transfection of MDA-MB-231 with an expression vector encoding ST6GaINAc I induces the expression of STn antigen at the cell surface. The expression of STn short cuts the initial O-glycosylation pattern of these cell lines, by competing with the Core-1 beta1,3-galactosyltransferase, the first enzyme involved in the elongation of O-glycan chains. Moreover, we show that STn expression is associated with morphological changes, decreased growth and increased migration of MDA-MB-231 cells.

The mechanisms of verapamil and tetraethylammonium (TEA) inhibition of voltage-gated K+ channels in LNCaP human prostate cancer cells were studied in whole-cell and outside/inside-out patch-clamp configurations. Rapidly activating outward K+ currents (I(K)) exhibited neither C-type, nor rapid (human ether á go-go-related gene-type) inactivation. With 2 mM [Mg(2+)](o), I(K) activation kinetics was independent of holding potential, suggesting the absence of ether á go-go-type K+ channels. Extracellular applications of TEA and verapamil (IC(50) = 11 microM) rapidly (12 s) inhibited I(K) in LNCaP cells. Blocking was also rapidly reversible. Intracellular TEA (1 mM), verapamil (1 mM), and membrane-impermeable N-methyl-verapamil (25 microM) did not influence whole-cell I(K), although both phenylalkylamines inhibited single-channel currents in inside-out patches. Extracellular application of N-methyl-verapamil (25 microM) had no influence on I(K). Our results are compatible with the hypothesis that, in LNCaP cells expressing C-type inactivation-deficient voltage-activated K+ channels, phenylalkylamines interact with an intracellular binding site, and probably an additional hydrophobic binding site that does not bind charged phenylalkylamines. The inhibiting effects of verapamil and TEA on I(K) were additive, suggesting independent K+-channel blocking mechanisms. Indeed, TEA (1 mM) reduced a single-channel conductance (from 7.3 +/- 0.5 to 3.2 +/- 0.4 pA at a membrane potential of +50 mV, n = 6), whereas verapamil (10 microM) reduced an open-channel probability (from 0.45 +/- 0.1 in control to 0.1 +/- 0.09 in verapamil-treated cells, n = 9). The inhibiting effects of verapamil and TEA on LNCaP cell proliferation were not multiplicative, suggesting that both share a common antiproliferative mechanism initiated through a K+ channel block.

The class of molecular chaperones known as 14-3-3 is involved in the control of cellular growth by virtue of its apparent regulation of various signaling pathways, including the Raf/mitogen-activated protein kinase pathway. In breast cancer cells, the sigma form of 14-3-3 has been shown to interact with cyclin-dependent kinases and to control the rate of entry into mitosis. To test for a direct role for 14-3-3 in breast epithelial cell neoplasia, we have quantitated 14-3-3 protein levels using a proteomic approach based on two-dimensional electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF). We show here that 14-3-3sigma protein is strongly down-regulated in the prototypic breast cancer cell lines MCF-7 and MDA-MB-231 and in primary breast carcinomas as compared with normal breast epithelial cells. In contrast, levels of the alpha, beta, delta, or zeta isoforms of 14-3-3 were the same in both normal and transformed cells. The data support the idea that 14-3-3sigma is involved in the neoplastic transition of breast epithelial cells by virtue of its role as a tumor suppressor; as such, it may constitute a robust marker with clinical efficacy for this pathology.

MCF-7 cells express voltage-activated K+ channels. In the present study, we used the patch-clamp and RT-PCR techniques to investigate the involvement of these channels during the cell cycle progression. The outward rectifier current (IK) recorded during depolarization was almost completely suppressed by the classical K+ channel blocker tetraethylammonium (TEA) in MCF-7 cells. TEA also inhibited cell proliferation, as measured with 3H-thymidine incorporation. Moreover, profound changes were observed in both the resting membrane potential (RMP) and IK during the release from the G0/G1 phase of the cell cycle. MCF-7 cells arrested in G0/G1 were depolarized (-26.3 +/- 10 mV, n = 30) and IK-density was small (9.4 +/- 5.6 pA/pF, n = 60) compared to cells progressing in the G1 phase (RMP = -60 +/- 7.9 mV; n = 35 and IK-density = 30.2 +/- 8.5 pA/pF; n = 76). IK was highly sensitive to Mg2+, astemizole and TEA (10 mM). Extracellular perfusion of 5 mM Mg2+ dramatically slowed the activation and perfusion of 2 microM astemizole inhibited both IK (20 +/- 3%) and cell proliferation (23%). Moreover, the h-EAG mRNA expression was modulated during the cell cycle. Thus, these data suggested that h-EAG K+ channels play a role in controlling the proliferation and/or cell cycle.

Fibroblast growth factor-2 (FGF-2) is a potent regulator of breast cancer cell growth through stimulation of tyrosine kinase receptors and activation of the mitogen-activated protein kinase cascade. In the present study, we have investigated changes in protein synthesis induced by FGF-2 stimulation of the prototypic human breast cancer cell line MCF-7. Using high-resolution two-dimensional electrophoresis of 35S amino acid metabolically labeled proteins and computerized analysis of 2D autoradiograms, we found that four proteins were up-regulated within the first 12 h of FGF-2 stimulation. Mass spectrometry analysis (MALDI-TOF and MS-MS) of tryptic fragments and database searches allowed the identification of these FGF-2-regulated proteins as the heat shock proteins HSP90 and HSP70, the proliferating cell nuclear antigen (PCNA), and the transcriptionaly controlled tumor protein (TCTP). We then analyzed the distribution of these proteins in various cancerous and normal breast epithelial cells. Interestingly, the four FGF-2-regulated proteins were found to be constitutively up-regulated in ras-transfected MCF-7 cells, indicating their relevance to the up-regulation of cellular proliferation. Moreover, HSP90 and PCNA were found at higher levels in cancerous cells than in normal cells. The role of HSP90 was further investigated using the specific inhibitor geldanamycin. We showed that the functionality of HSP90 is strictly required in order to obtain FGF-2 mitogenic stimulation in MCF-7 cells, indicating the crucial role played by this molecular chaperone in the control of breast cancer cell growth. Finally, these results show that proteomic analysis is a valuable method for identifying potential markers or therapeutic targets related to cancer growth.

We show here that the neurotrophin nerve growth factor (NGF), which has been shown to be a mitogen for breast cancer cells, also stimulates cell survival through a distinct signaling pathway. Breast cancer cell lines (MCF-7, T47-D, BT-20, and MDA-MB-231) were found to express both types of NGF receptors: p140(trkA) and p75(NTR). The two other tyrosine kinase receptors for neurotrophins, TrkB and TrkC, were not expressed. The mitogenic effect of NGF on breast cancer cells required the tyrosine kinase activity of p140(trkA) as well as the mitogen-activated protein kinase (MAPK) cascade, but was independent of p75(NTR). I, contrast, the anti-apoptotic effect of NGF (studied using the ceramide analogue C2) required p75(NTR) as well as the activation of the transcription factor NF-kB, but neither p140(trkA) nor MAPK was necessary. Other neurotrophins (BDNF, NT-3, NT-4/5) also induced cell survival, although not proliferation, emphasizing the importance of p75(NTR) in NGF-mediated survival. Both the pharmacological NF-KB inhibitor SN50, and cell transfection with IkBm, resulted in a diminution of NGF anti-apoptotic effect. These data show that two distinct signaling pathways are required for NGF activity and confirm the roles played by p75(NTR) and NF-kappaB in the activation of the survival pathway in breast cancer cells.

The effects of the polypeptide hormone prolactin (PRL) in the development and regulation of benign prostate hyperplasia (BPH) and also in prostate cancer are not very well characterized. This study examines the action of PRL, either alone or in association with androgens [testosterone (T) or dihydrotestosterone (DHT)], in the rat prostate gland. The effects of PRL and androgens were investigated after 30 and 60 days in control, castrated, castrated with a substitutive implant of T or DHT, and sham-operated Wistar rats. To enhance PRL release, we induced hyperprolactinemia by administering chronic injections of sulpiride (40 mg. kg(-1). day(-1)). Chronic hyperprolactinemia induces enlargement and inflammation of the lateral rat prostate without any histological changes on ventral and dorsal lobes. We also demonstrate that hyperprolactinemia induces Bcl-2 overexpression in the lateral rat prostate and that this could inhibit the level of apoptosis. The in vivo model established here is a useful in vivo approach for studying the hormonal regulation of normal and pathological prostate development.

1. In the present study, we investigated the mechanisms involved in the induction of apoptosis by the Ca2+-ATPase inhibitor thapsigargin (TG), in androgen-sensitive human prostate cancer LNCaP cells. 2. Exposure of fura-2-loaded LNCaP cells to TG in the presence of extracellular calcium produced an increase in intracellular Ca2+, the first phase of which was associated with depletion of intracellular stores and the second one with consecutive extracellular Ca2+ entry through plasma membrane, store-operated Ca2+ channels (SOCs). 3. For the first time we have identified and characterized the SOC-mediated membrane current (Istore) in prostate cells using whole-cell, cell-attached, and perforated patch-clamp techniques, combined with fura-2 microspectrofluorimetric and Ca2+-imaging measurements. 4. Istore in LNCaP cells lacked voltage-dependent gating and displayed an inwardly rectifying current-voltage relationship. The unitary conductance of SOCs with 80 mM Ca2+ as a charge carrier was estimated at 3.2 +/- 0.4 pS. The channel has a high selectivity for Ca2+ over monovalent cations and is inhibited by Ni2+ (0.5-3 mM) and La3+ (1 microM). 5. Treatment of LNCaP cells with TG (0.1 microM) induced apoptosis as judged from morphological changes. Decreasing extracellular free Ca2+ to 200 nM or adding 0.5 mM Ni2+ enhanced TG-induced apoptosis. 6. The ability of TG to induce apoptosis was not reduced by loading the cells with intracellular Ca2+ chelator (BAPTA-AM). 7. These results indicate that in androgen-sensitive prostate cancer cells the depletion of intracellular Ca2+ stores may trigger apoptosis but that there is no requirement for the activation of store-activated Ca2+ current and sustained Ca2+ entry in induction and development of programmed cell death.

FGFs are pleiotropic growth factors that control cell proliferation, migration and differentiation. However, FGF transduction studies have so far focused primarily on the mitogenic effect of this growth factor family and it has been difficult to assess if the described intracellular signaling pathways are dedicated solely to cell proliferation, or whether they are equally important for the migratory activity often seen in responsive cells. We review here papers in which the migratory effects of this growth factor family were clearly discriminated from proliferative effects. In toto, these studies suggest that cells use different signaling pathways for migration, such as Src and p38 MAP kinase, from those for proliferation, which tend to upregulate the ERKs. Which signaling pathway a cell uses for proliferation or migration appears to depend on many factors, including the structure and the quantity of available FGF trapped in the basal lamina by heparan sulfate co-factors, the disposition of cognate high affinity receptors and the general environment of the cell. Thus the density of the cell population, the state of the cell cycle, the presence of other factors or receptors will modulate the migratory response of cells to FGF.

BACKGROUND. The growth of the prostate gland is mainly dependent on androgens. Other hormones, like prolactin (PRL), also influence prostate development. Our purpose was to analyze and compare the effects of two drugs (5 alpha-reductase inhibitor) used in the therapy of benign prostatic hyperplasia: lipidosterolic extract of Serenoa repens (LSESR), and finasteride in an in vivo model of rat prostate hyperplasia induced by hyperprolactinemia.METHODS. Hyperprolactinemia was induced by 30 daily injections of sulpiride. Wistar rats received daily gavages of LSESR or finasteride. We used the following groups: control, castrated, castrated with a substitute testosterone (T), or 5 alpha-dihydrotestosterone (DHT) implant.RESULTS. Hyperprolactinemia increases the wet weight and induces hyperplasia in the lateral prostate (LP). Unlike finasteride, LSESR significantly reduced LP growth and hyperplasia in castrated, DHT-implanted, and sulpiride-treated rats.CONCLUSIONS. Finasteride was only capable of inhibiting the effect of androgens on rat prostate enlargement. LSESR inhibited not only the androgenic but also the trophic effect of PRL in rat LP hyperplasia. Prostate 43:49-58, 2000. (C) 2000 Wiley-Liss, Inc.