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1.  Wip1 sensitizes p53-negative tumors to apoptosis by regulating the Bax/Bcl-xL ratio 
Cell Cycle  2012;11(10):1883-1887.
Wip1 is a stress-response phosphatase that negatively regulates several tumor suppressors, including p53. In a sizeable fraction of tumors, overexpression or amplification of Wip1 compromises p53 functions; inhibition of Wip1 activity is an attractive strategy for improving treatment of these tumors. However, over half of human tumors contain mutations in the p53 gene or have lost both alleles. Recently, we observed that in cancer cells lacking wild type p53, reduction of Wip1 expression was ineffective, whereas, surprisingly, overexpression of Wip1 increased anticancer drug sensitivity. The increased sensitivity resulted from activation of the intrinsic pathway of apoptosis through increased levels of the pro-apoptotic protein Bax and decreased levels of the anti-apoptotic protein Bcl-xL. We showed that interaction of Wip1 and the transcription factor RUNX2, specifically through dephosphorylation of RUNX2 phospho-S432, resulted in increased expression of Bax. Interestingly, overexpression of Wip1 increased drug sensitivity only in the p53-negative tumor cells while protecting the wild type p53-containing normal cells from drug-induced collateral injury. Here, we provide evidence that Wip1 overexpression decreases expression of Bcl-xL through negative regulation of NFκB activity. Thus, Wip1 overexpression increases the sensitivity of p53-negative cancer cells to anticancer drugs by separately affecting Bax and Bcl-xL protein levels.
PMCID: PMC3359118  PMID: 22544321
Bax; Bcl-xL; cisplatin; NFκB; p53-negative; phosphatase; Runx2; Wip1
2.  In vitro maturation, fertilization, embryo development & clinical outcome of human metaphase-I oocytes retrieved from stimulated intracytoplasmic sperm injection cycles 
Background & objectives:
The major cause of fertilisation failure after ICSI is failure of the oocyte to initiate the biochemical processes necessary for activation. This inability could be ascribed to cytoplasmic immaturity of those gametes even if they had reached nuclear maturity. The activation of a mature oocyte is characterised by release from metaphase II (MII) arrest and extrusion of the second polar body, followed by pro-nuclear formation. The aim of this study was to evaluate the fate of in vitro matured (IVM) metaphase I (MI) oocytes subjected to intracytoplasmic sperm injection (ICSI) at different time intervals after extrusion of the first polar body (1PB) in in vitro fertilization (IVF) cycles.
A total of 8030 oocytes were collected from 1400 ICSI cycles, 5504 MII at the time of cumulus retrieval. Four hundred eight metaphase II (MII) (27.1%) matured to MII after in vitro culture for 2-26 h and 5389 sibling MII in the moment of oocyte denudation were injected. On the other hand, 49 ICSI cycles containing only MI oocytes at retrieval were injected at three different time intervals after reaching the MII. The intervals were as follows: 2-6 h (n=10), 8-11 h (n=4) and 23-26 h (n=10). Fertilization and development potential were evaluated in both studies.
Fertilization, embryo cleavage and quality were significantly lower in IVM MI compared to MII at time of denudation. Pregnancy rate was higher in group MII. Pregnancy was achieved in three embryo transfers when ICSI was performed within 2-6 h (group I) and 8-11 h (group II) after PB extrusion. One pregnancy was obtained in group I and a healthy neonate was born.
Interpretation & conclusions:
Immature oocytes from women whose ovaries have been stimulated could be matured, fertilized by ICSI, cleaved in vitro and to give rise to a live birth. However, the developmental competence of embryos derived from immature oocytes is reduced, compared with sibling in vivo matured oocytes. Further, human IVM oocytes need between 2-6h after the 1PB extrusion to complete its maturation.
PMCID: PMC3657857  PMID: 23563377
Controlled ovarian hyperstimulated cycle; ICSI timing; in vitro maturation; first polar body; metaphase-I
3.  Chemotherapy overcomes TRAIL-R4-mediated TRAIL resistance at the DISC level 
Cell Death and Differentiation  2010;18(4):700-711.
Apo2L/TRAIL is a promising anti-cancer drug owing to its ability to trigger apoptosis by binding to TRAIL-R1 or TRAIL-R2, two membrane bound receptors that are often expressed by tumor cells. TRAIL can also bind non-functional receptors such as TRAIL-R4, but controversies still exist regarding their potential to inhibit TRAIL-induced apoptosis.
We show here that TRAIL-R4, expressed either endogenously or ectopically, inhibits TRAIL induced apoptosis. Interestingly, the combination of chemotherapeutic drugs with TRAIL restores tumor cell sensitivity to apoptosis in TRAIL-R4 expressing cells. This sensitization, which mainly occurs at the DISC level, through enhanced caspase-8 recruitment and activation, is compromised by c-FLIP expression and is independent of the mitochondria.
Importantly, TRAIL-R4 expression prevents TRAIL-induced tumor regression in nude mice, but tumor regression induced by TRAIL can be restored with chemotherapy.
Our results clearly support a negative regulatory function for TRAIL-R4 in controlling TRAIL signaling, and unveil TRAIL-R4’s ability to cooperate with c-FLIP to inhibit TRAIL-induced cell death.
PMCID: PMC3117243  PMID: 21072058
TRAIL; chemotherapy; cancer
4.  Implication of Heat Shock Factors in Tumorigenesis: Therapeutical Potential 
Cancers  2011;3(1):1158-1181.
Heat Shock Factors (HSF) form a family of transcription factors (four in mammals) which were named according to the discovery of their activation by a heat shock. HSFs trigger the expression of genes encoding Heat Shock Proteins (HSPs) that function as molecular chaperones, contributing to establish a cytoprotective state to various proteotoxic stresses and in pathological conditions. Increasing evidence indicates that this ancient transcriptional protective program acts genome-widely and performs unexpected functions in the absence of experimentally defined stress. Indeed, HSFs are able to re-shape cellular pathways controlling longevity, growth, metabolism and development. The most well studied HSF, HSF1, has been found at elevated levels in tumors with high metastatic potential and is associated with poor prognosis. This is partly explained by the above-mentioned cytoprotective (HSP-dependent) function that may enable cancer cells to adapt to the initial oncogenic stress and to support malignant transformation. Nevertheless, HSF1 operates as major multifaceted enhancers of tumorigenesis through, not only the induction of classical heat shock genes, but also of “non-classical” targets. Indeed, in cancer cells, HSF1 regulates genes involved in core cellular functions including proliferation, survival, migration, protein synthesis, signal transduction, and glucose metabolism, making HSF1 a very attractive target in cancer therapy. In this review, we describe the different physiological roles of HSFs as well as the recent discoveries in term of non-cogenic potential of these HSFs, more specifically associated to the activation of “non-classical” HSF target genes. We also present an update on the compounds with potent HSF1-modulating activity of potential interest as anti-cancer therapeutic agents.
PMCID: PMC3756408  PMID: 24212658
Heat Shock Factors; cancer; therapeutical approaches
5.  Heat Shock Proteins as Danger Signals for Cancer Detection 
First discovered in 1962, heat shock proteins (HSPs) are highly studied with about 35,500 publications on the subject to date. HSPs are highly conserved, function as molecular chaperones for a large panel of “client” proteins and have strong cytoprotective properties. Induced by many different stress signals, they promote cell survival in adverse conditions. Therefore, their roles have been investigated in several conditions and pathologies where HSPs accumulate, such as in cancer. Among the diverse mammalian HSPs, some members share several features that may qualify them as cancer biomarkers. This review focuses mainly on three inducible HSPs: HSP27, HPS70, and HSP90. Our survey of recent literature highlights some recurring weaknesses in studies of the HSPs, but also identifies findings that indicate that some HSPs have potential as cancer biomarkers for successful clinical applications.
PMCID: PMC3355996  PMID: 22649762
heat shock protein; danger signal; detection; biomarker; stress; cancer
6.  Membrane-associated Hsp72 from tumor-derived exosomes mediates STAT3-dependent immunosuppressive function of mouse and human myeloid-derived suppressor cells 
Myeloid-derived suppressor cells (MDSCs) have been identified in humans and mice as a population of immature myeloid cells with the ability to suppress T cell activation. They accumulate in tumor-bearing mice and humans and have been shown to contribute to cancer development. Here, we have isolated tumor-derived exosomes (TDEs) from mouse cell lines and shown that an interaction between TDE-associated Hsp72 and MDSCs determines the suppressive activity of the MDSCs via activation of Stat3. In addition, tumor-derived soluble factors triggered MDSC expansion via activation of Erk. TDE-associated Hsp72 triggered Stat3 activation in MDSCs in a TLR2/MyD88-dependent manner through autocrine production of IL-6. Importantly, decreasing exosome production using dimethyl amiloride enhanced the in vivo antitumor efficacy of the chemotherapeutic drug cyclophosphamide in 3 different mouse tumor models. We also demonstrated that this mechanism is relevant in cancer patients, as TDEs from a human tumor cell line activated human MDSCs and triggered their suppressive function in an Hsp72/TLR2-dependent manner. Further, MDSCs from cancer patients treated with amiloride, a drug used to treat high blood pressure that also inhibits exosome formation, exhibited reduced suppressor functions. Collectively, our findings show in both mice and humans that Hsp72 expressed at the surface of TDEs restrains tumor immune surveillance by promoting MDSC suppressive functions.
PMCID: PMC2810085  PMID: 20093776
7.  Apoptosis Versus Cell Differentiation 
Prion  2007;1(1):53-60.
Heat shock proteins HSP27, HSP70 and HSP90 are molecular chaperones whose expression is increased after many different types of stress. They have a protective function helping the cell to cope with lethal conditions. The cytoprotective function of HSPs is largely explained by their anti-apoptotic function. HSPs have been shown to interact with different key apoptotic proteins. As a result, HSPs can block essentially all apoptotic pathways, most of them involving the activation of cystein proteases called caspases. Apoptosis and differentiation are physiological processes that share many common features, for instance, chromatin condensation and the activation of caspases are frequently observed. It is, therefore, not surprising that many recent reports imply HSPs in the differentiation process. This review will comment on the role of HSP90, HSP70 and HSP27 in apoptosis and cell differentiation. HSPs may determine de fate of the cells by orchestrating the decision of apoptosis versus differentiation.
PMCID: PMC2633709  PMID: 19164900
apoptosis; differentiation; heat shock proteins; chaperones; cancer cells; anticancer drugs
8.  Caspase-dependent immunogenicity of doxorubicin-induced tumor cell death 
The Journal of Experimental Medicine  2005;202(12):1691-1701.
Systemic anticancer chemotherapy is immunosuppressive and mostly induces nonimmunogenic tumor cell death. Here, we show that even in the absence of any adjuvant, tumor cells dying in response to anthracyclins can elicit an effective antitumor immune response that suppresses the growth of inoculated tumors or leads to the regression of established neoplasia. Although both antracyclins and mitomycin C induced apoptosis with caspase activation, only anthracyclin-induced immunogenic cell death was immunogenic. Caspase inhibition by Z-VAD-fmk or transfection with the baculovirus inhibitor p35 did not inhibit doxorubicin (DX)-induced cell death, yet suppressed the immunogenicity of dying tumor cells in several rodent models of neoplasia. Depletion of dendritic cells (DCs) or CD8+T cells abolished the immune response against DX-treated apoptotic tumor cells in vivo. Caspase inhibition suppressed the capacity of DX-killed cells to be phagocytosed by DCs, yet had no effect on their capacity to elicit DC maturation. Freshly excised tumors became immunogenic upon DX treatment in vitro, and intratumoral inoculation of DX could trigger the regression of established tumors in immunocompetent mice. These results delineate a procedure for the generation of cancer vaccines and the stimulation of anti-neoplastic immune responses in vivo.
PMCID: PMC2212968  PMID: 16365148
9.  HSP27 Is a Ubiquitin-Binding Protein Involved in I-κBα Proteasomal Degradation 
Molecular and Cellular Biology  2003;23(16):5790-5802.
HSP27 is an ATP-independent chaperone that confers protection against apoptosis through various mechanisms, including a direct interaction with cytochrome c. Here we show that HSP27 overexpression in various cell types enhances the degradation of ubiquitinated proteins by the 26S proteasome in response to stressful stimuli, such as etoposide or tumor necrosis factor alpha (TNF-α). We demonstrate that HSP27 binds to polyubiquitin chains and to the 26S proteasome in vitro and in vivo. The ubiquitin-proteasome pathway is involved in the activation of transcription factor NF-κB by degrading its main inhibitor, I-κBα. HSP27 overexpression increases NF-κB nuclear relocalization, DNA binding, and transcriptional activity induced by etoposide, ΤNF-α, and interleukin 1β. HSP27 does not affect I-κBα phosphorylation but enhances the degradation of phosphorylated I-κBα by the proteasome. The interaction of HSP27 with the 26S proteasome is required to activate the proteasome and the degradation of phosphorylated I-κBα. A protein complex that includes HSP27, phosphorylated I-κBα, and the 26S proteasome is formed. Based on these observations, we propose that HSP27, under stress conditions, favors the degradation of ubiquitinated proteins, such as phosphorylated I-κBα. This novel function of HSP27 would account for its antiapoptotic properties through the enhancement of NF-κB activity.
PMCID: PMC166315  PMID: 12897149
10.  Caspase Activation Is Required for Terminal Erythroid Differentiation 
The cysteine proteases known as caspases play a central role in most apoptotic pathways. Here, we show that caspase inhibitors arrest the maturation of human erythroid progenitors at early stages of differentiation, before nucleus and chromatin condensation. Effector caspases such as caspase-3 are transiently activated through the mitochondrial pathway during erythroblast differentiation and cleave proteins involved in nucleus integrity (lamin B) and chromatin condensation (acinus) without inducing cell death and cleavage of GATA-1. These observations indicate a new function for caspases as key proteases in the process of erythroid differentiation.
PMCID: PMC2193347  PMID: 11208865
apoptosis; erythropoiesis; mitochondria; acinus; lamin B
11.  The Viral Nucleocapsid Protein of Transmissible Gastroenteritis Coronavirus (TGEV) Is Cleaved by Caspase-6 and -7 during TGEV-Induced Apoptosis 
Journal of Virology  2000;74(9):3975-3983.
The transmissible gastroenteritis coronavirus (TGEV), like many other viruses, exerts much of its cytopathic effect through the induction of apoptosis of its host cell. Apoptosis is coordinated by a family of cysteine proteases, called caspases, that are activated during apoptosis and participate in dismantling the cell by cleaving key structural and regulatory proteins. We have explored the caspase activation events that are initiated upon infection of the human rectal tumor cell line HRT18 with TGEV. We show that TGEV infection results in the activation of caspase-3, -6, -7, -8, and -9 and cleavage of the caspase substrates eIF4GI, gelsolin, and α-fodrin. Surprisingly, the TGEV nucleoprotein (N) underwent proteolysis in parallel with the activation of caspases within the host cell. Cleavage of the N protein was inhibited by cell-permeative caspase inhibitors, suggesting that this viral structural protein is a target for host cell caspases. We show that the TGEV nucleoprotein is a substrate for both caspase-6 and -7, and using site-directed mutagenesis, we have mapped the cleavage site to VVPD359↓. These data demonstrate that viral proteins can be targeted for destruction by the host cell death machinery.
PMCID: PMC111911  PMID: 10756009
12.  New insights into the kinetic resistance to anticancer agents 
Cytotechnology  1998;27(1-3):225-235.
Kinetic resistance plays a major role in the failure of chemotherapy towards many solid tumors. Kinetic resistance to cytotoxic drugs can be reproduced in vitro by growing the cells as multicellular spheroids (Multicellular Resistance) or as hyperconfluent cultures (Confluence-Dependent Resistance). Recent findings on the cell cycle regulation have permitted a better understanding why cancer cells which arrest in long quiescent phases are poorly sensitive to cell-cycle specific anticancer drugs. Two cyclin-dependent kinase inhibitors (CDKI) seem particularly involved in the cell cycle arrest at the G1 to S transition checkpoint: the p53-dependent p21cip1 protein which is activated by DNA damage and the p27kip1 which is a mediator of the contact inhibition signal. Cell quiescence could alter drug-induced apoptosis which is partly dependent on an active progression in the cell cycle and which is facilitated by overexpression of oncogenes such as c-Myc or cyclins. Investigations are yet necessary to determine the influence of the cell cycle on the balance between antagonizing (bcl-2, bcl-XL...) or stimulating (Bax, Bcl-XS, Fas...) factors in chemotherapy-induced apoptosis. Quiescent cells could also be protected from toxic agents by an enhanced expression of stress proteins, such as HSP27 which is induced by confluence. New strategies are required to circumvent kinetic resistance of solid tumors: adequate choice of anticancer agents whose activity is not altered by quiescence (radiation, cisplatin), recruitment from G1 to S/G2 phases by cell pretreatment with alkylating drugs or attenuation of CDKI activity by specific inhibitors.
PMCID: PMC3449571  PMID: 19002794
anticancer drugs; apoptosis; cell cycle; drug resistance

Results 1-12 (12)