Notch signaling pathway controls key functions in vascular and endothelial cells (ECs) where Notch4 plays a major role. However, little is known about the contribution of other Notch receptors. This study investigated regulation of Notch2 and further examined its implication in EC dysfunction.Here, we provide evidence for a novel link between Notch and TNF signaling, where Notch2 is upregulated and activated in response to TNF. Forced expression of Notch2 intracellular domain in cultured ECs promotes apoptosis and allows the significant downregulation of several cell-death-related transcripts in a dose-dependent manner. In particular, activation of Notch2 led to a rapid decrease in survivin mRNA and protein expression, while survivin upregulation was obtained by the selective knockdown of Notch2 in ECs, indicating that survivin expression is controlled at the Notch level. Moreover, Notch2 silencing and ectopic expression of survivin, but not XIAP or Bcl2, rescued ECs from TNF and Notch2-mediated apoptosis, respectively.In conclusion, TNF signaling activates Notch2 that sensitizes ECs to apoptosis via modulation of the key apoptosis regulator survivin. Overall, our findings also indicate that specific Notch receptors control distinct functions in vascular cells and inflammatory cytokines contribute to this specificity.
La pénurie d'organes disponibles est responsable d'un nombre croissant de décès de patients en attente de greffe. Dans ces conditions, des solutions alternatives sont envisagées, notamment l'utilisation d'organes porcins. Les xénogreffes sont cependant soumises à différents types de rejet (hyperaigu, vasculaire aigu et cellulaire) dont les mécanismes commencent à être mieux connus. Les stratégies visant à faire de la xénotransplantation une réalité clinique existent et, bien que le nombre de laboratoires engagés dans ce domaine soit encore modeste, des progrès substantiels ont été récemment obtenus. Elles incluent de nouvelles modifications génétiques des animaux donneurs combinées à la mise au point de traitements immunosuppresseurs adaptés à la situation clinique, ainsi que les tentatives d'induction d'une tolérance immunitaire et la gestion du risque sanitaire. L'acceptation des xénogreffes par les receveurs potentiels et son impact sur la société sont également pris en considération.
Survivin is selectively expressed in most of common human cancers and is now viewed as a potent modulator of the cell death/proliferation balance in tumour cells. We previously found that myeloma cells expressed high levels of Survivin protein in correlation with disease progression and that Survivin knock-down by RNA interference decreased myeloma cell growth. We now demonstrate that Survivin overexpression promotes the proliferation and survival of human myeloma cells both in vitro and in vivo in the absence of their major growth factor, interleukin 6. Of particular interest, this effect correlates with the down regulation of Bim, a critical BH3-only cell death activator during cytokine deprivation, mainly at transcriptional level. The tight link between Survivin and Bim expression, reported for the first time here in myeloma cells and in other cell lines, is further confirmed in a panel of newly diagnosed patients with myeloma, and BIRC5 is validated as a gene significantly associated with short survival in these patients. Altogether, our findings provide evidence that Survivin directly contributes to malignant progression of myeloma and strongly suggest that targeting Survivin may disrupt the delicate balance controlling cell survival and proliferation, opening new avenues for the therapy of this still difficult-to-treat cancer.
Abstract INTRODUCTION Among breast cancers, the triple negative subtype (negative for hormone receptors and not overexpressing HER2) has the worst prognosis and its response to Iniparib has been investigated in clinical trials. Further investigations are needed to optimize drug schedule and patient selection criteria. Iniparib antitumor mechanism is not completely understood, as well as iniparib diffusion kinetic into tumoral tissues. We address these questions in spontaneous canine invasive mammary carcinomas, which are a good model for this cancer subtype (Ibisch et al., World Veterinary Cancer Congress 2012), in a neoadjuvant setting. To our knowledge, this is the first study of iniparib administration in cancer-bearing dogs. MATERIAL AND METHODS Twenty female dogs with spontaneous MT with malignant criteria (tumor size, speed of growth, ulceration, relapse, or metastasis) were included. All tumors were described as rapidly growing. Dogs received a first infusion of iniparib at day 0 and a combination of carboplatin and iniparib at day 7. Biological materials (tumor biopsies and blood) were collected before and 5 minutes after iniparib infusion for pharmacokinetic and metabolism studies. Tumor response was evaluated by caliper measurements and histopathological analysis of mammary tumors and draining lymph nodes. A chain mastectomy was performed 3 or 4 weeks later. Histological records included the subtype of carcinoma (WHO 1999), Elston & Ellis grade, presence of emboli, lymph node metastasis and IHC stainings using ER, PR, Her2 (scored according to Wolff et al.2007), CK5/6, EGF-R and Ki67. Intensity of necrosis and apoptosis was evaluated using PAS coloration and immunohistochemistry for caspase 3, at DO on tumor biopsies and at surgery. Toxicity of the protocol was evaluated and its efficiency on invasive carcinomas was compared to surgery alone (control group of 27 female dogs with invasive mammary carcinomas treated by chain mastectomy alone). RESULTS Treated and control groups shared similar features concerning animal breeds, age, neutering status and tumor location. 75% of the treated MT were malignant. Necrosis and apoptosis were significantly increased in respectively 63 and 56% of iniparib treated tumors. Clinical evidence of toxicity was minimal (15% of dogs with nausea, 60% with transient polyuria-polydipsia). Tumor stabilization was observed before surgery in all dogs but one. Median survival has not been reached. CONCLUSION Iniparib at 35mg/kg combined with carboplatin at 300 mg/m2 seemed well tolerated in this study and deserves further investigations. The degree of necrosis and apoptosis in the treated tumors can be evaluated with these techniques. Iniparib pharmacokinetic and metabolism studies in cancer-bearing dogs are ongoing. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-05-08.
Découverte il y a dix ans, Survivine orchestre à la fois le cycle et la mort cellulaire, deux fonctions particulièrement dérégulées dans les cellules cancéreuses. Présente au cours du développement embryonnaire, puis absente des tissus différenciés adultes, Survivine est fortement exprimée dans la plupart des tumeurs. Sa capacité à interagir avec différents partenaires moléculaires et sa distribution subcellulaire commencent à être mieux connues. Ses propriétés pro-oncogéniques multiples et sa valeur pronostique démontrée dans divers cancers ont fait de Survivine une cible anti-tumorale particulièrement attractive et les premiers essais cliniques semblent prometteurs.
P1037 Aims: Blockade of individual T cell costimulatory pathways may result in long-term allograft survival but tolerance is not obtained since chronic rejection is observed. To obtain transplantation tolerance, blockade of more than one costimulatory pathway is needed but the importance of different pathways and the optimal reagents to be used have not been fully defined. Methods: Heart transplantation was performed between MHC mismatched rats. CD40-CD154 interactions were blocked by gene transfer of CD40Ig. We interfered with CD28 costimulatory signals by administering an anti-CD28 Mab, which in contrast to CTLA4Ig, preserves T cell inhibitory signals through CTLA4. Results: CD40Ig treatment resulted in long-term (>120d, n=19) heart allograft survival and development of chronic rejection in all grafts whereas acute rejection occurred in the non-coding adenovirus-treated (7.6±1.6d, n=9) group. Treatment with anti-CD28 Mab resulted in a moderate but significant prolongation of allograft survival (17.4±1.7d, n=10). Cotreatment with both CD40Ig and anti-CD28Mab resulted in long term allograft survival (>120d, n=7) and a significant reduction in the percentage and degree of vessels with chronic rejection lesions, with 3/7 grafts completely free of lesions and therefore tolerant. Splenocytes from cotreated tolerant recipients, but not those with chronic rejection (CD40Ig or cotreated), showed completely suppressed MLR responses against 1st but not 3rd party donor cells. T cells purified from splenocytes of tolerant cotreated recipients showed normal MLR responses indicating that among total splenocytes, non T cells inhibited the proliferation of T cells. Splenocytes from tolerant recipients but not those with chronic rejection lesions inhibited naïve MLR, indicating the presence of active tolerogenic mechanisms. Transfer of splenocytes from CD40Ig-treated recipients to naïve sublethaly irradiated rats conferred long term allograft survival (>120d, n=4 vs. 17 d, n=2 in controls). In contrast, transfer of splenocytes from CD40Ig+anti-CD28 cotreated rats, either tolerant or with chronic rejection lesions, did not prolong allograft survival (17d, n=3). Conclusions: The association of CD40Ig with anti-CD28 Mab resulted in decreased chronic rejection lesions and tolerogenic mechanisms different to those seen with CD40Ig.
: Blockade of the CD40-CD40L pathway results in long-term allograft survival but does not prevent chronic rejection. ICOS-ICOSL are members of the CD28-B7 family that play an important role in T-cell activation.: The authors analyzed the effect of single or combined treatment with an anti-ICOS monoclonal antibody and CD40 immunoglobulin (Ig) on acute and chronic rejection of heart allografts in rats.: Treatment with anti-ICOS resulted in a modest but significant prolongation of allograft survival. Treatment with CD40Ig resulted in long-term graft survival but the cardiac grafts developed chronic rejection lesions. Combined CD40Ig+anti-ICOS treatment led to indefinite graft survival in all recipients and a significant decrease of chronic rejection lesions compared with CD40Ig alone. Importantly, four of the seven CD40Ig+anti-ICOS-treated recipients showed a complete absence of chronic rejection lesions, whereas all of the CD40Ig-treated recipients showed chronic rejection. The CD40Ig+anti-ICOS group also showed significant decreased graft infiltration, decreased antidonor cytotoxic T-lymphocyte activity, and decreased alloantibodies compared with the CD40Ig-treated group. Adoptive transfer of splenocytes indefinitely prolonged allograft survival, whereas those depleted of T cells did not, suggesting the development of T-regulatory mechanisms.: These data indicate that the chronic rejection mechanisms that are CD40-CD40L independent are ICOS-ICOSL dependent. These results were obtained with conservation of cognate immune responses and development of tolerogenic T cells.
Abstract: Background: In heart allograft in the rat, a sustained costimulation blockade with CTLA4Ig prevents alloreactive T‐cell activation and promotes a long‐term graft survival through the action of tolerogeneic dendritic cells. It is unclear whether similar mechanisms might occur after xenotransplantation. To test that hypothesis, we have analyzed the action of CTLA4Ig in a model of CD4 + T cell‐mediated xenograft rejection. Methods: Hamster hearts were transplanted into LEW.1A rats receiving an accommodation‐inducing treatment consisting of a short course administration of LF15‐0195 and a daily administration of cyclosporine A (CSA). To achieve long‐term delivery of CTLA4Ig, an intravenous administration of an adenovirus vector coding for mouse CTLA4Ig (Ad‐CTLA4Ig) was added to the accommodation induction protocol. On day 40 post‐transplantation, rejection was induced by CSA withdrawal. In other xenograft recipients, CD28/B7 costimulation was inhibited at that time only by injections of CTLA4Ig or anti‐CD28 antibodies. Graft survival, immunohistology, as well as development of antibodies and regulatory cells were examined. Results: Xenografts survived 6 days after CSA withdrawal in controls and were rejected, as previously described, through the action of CD4 + xenoreactive T cells. Interfering with CD28/B7 costimulation inhibited this xenoreactive T cell response and delayed rejection to day 10. In recipients that had received Ad‐CTLA4Ig, survival was prolonged to day 19 and this was accompanied by the appearance of regulatory cells exhibiting non‐donor‐specific suppressive activity dependent on IL‐2, NO, and IDO. These regulatory cells were different from those previously identified after Ad‐CTLA4Ig administration in heart allograft in the rat. In these recipients, rejection occurred as a consequence of an evoked anti‐donor IgM response and complement activation and not of a cellular rejection as complement inhibition with cobra venom factor further prolonged xenograft survival. Conclusion: CD28/B7 blockade delays CD4 + T cell‐mediated rejection after CSA withdrawal in accommodated recipients of hamster heart xenografts. In addition, a sustained expression of CTLA4Ig has the potential of inducing cellular regulatory mechanisms. However, such treatment does not prevent the development of xenoreactive IgM antibodies that participate in vascular rejection processes in a complement‐dependent manner.
Abstract Blockade of CD40-CD40 ligand (CD40L) costimulation has been shown to synergize with that of CTLA4/CD28-B7 to promote transplant tolerance. To date, however, CD28-B7 interactions have been prevented using B7-blocking reagents like CTLA4-Ig that inhibit CD28-B7 together with CTLA4-B7 interactions. In this study, we have tested anti-CD28 Abs to prevent selectively CD28-B7 interactions while preserving CTLA4-B7 in addition to CD40-CD40L blockade. In the LEW.1W to LEW.1A rat combination, interfering with CD40-CD40L interactions by CD40Ig administration through gene transfer resulted in indefinite heart allograft survival due to the appearance of clonotypic CD8+CD45RClow regulatory T cells that were capable of transferring the tolerant state to naive animals. However, cardiac transplants in these recipients systematically developed chronic rejection lesions. Whereas anti-CD28 Ab monotherapy only delayed acute rejection and failed to induce tolerance, coadministration of anti-CD28 Abs and CD40Ig resulted in the long-term acceptation of allografts without chronic rejection lesions in 60% of the recipients, reduced the level of intragraft mRNA transcripts for cytokines and immune factors, and fully abrogated alloantibody production. In addition, the nature of regulatory cells was modified: the CD8+CD45RClow clonotypic T cells described in the CD40Ig-treated animals could not be found in cotreated animals, and the other CD8+CD45RClow cells had no regulatory activity and a different cytokine expression profile. Instead, in cotreated recipients we found IDO-dependent non-T cells with regulatory activity in vitro. Thus, the addition of a short-term anti-CD28 treatment with CD40Ig resulted in decreased heart allograft chronic rejection lesions, complete inhibition of Ab production, and modified regulatory mechanisms.
// Eloïse Véquaud 1 , Céline Séveno 1 , Delphine Loussouarn 1, 2 , Lucie Engelhart 1 , Mario Campone 1, 3 , Philippe Juin 1, 3 , Sophie Barillé-Nion 1 1 CRCNA, UMR INSERM U892, CNRS 6299, Université de Nantes, Team 8 « Cell Survival and Tumor Escape in Breast Cancers », Institut de Recherche en Santé de l'Université de Nantes, Nantes, France 2 Service d'Anatomie Pathologique, HGRL, CHU, Nantes University, Nantes, France 3 Institut de Cancérologie de Nantes, Centre de lutte contre le Cancer René Gauducheau, Boulevard Jacques Monod, Nantes, France Correspondence to: Sophie Barillé-Nion, e-mail: sophie.barille@inserm.fr Keywords: breast cancer, therapy, ex vivo assay Received: December 09, 2014 Accepted: April 25, 2015 Published: May 06, 2015 ABSTRACT Specific overexpression in cancer cells and evidence of oncogenic functions make Survivin an attractive target in cancer therapy. The small molecule compound YM155 has been described as the first "Survivin suppressant" but molecular mechanisms involved in its biological activity and its clinical potential remain obscure. We herein show that YM155 exerts single agent toxicity on primary breast cancer cells grown in an ex vivo assay preserving tumor microenvironment. In vitro assays indicate that YM155 more efficiently triggers cell death in breast cancer cells (including these with stem-cell like properties) than in non tumorigenic mammary cells. YM155-induced cell death is critically dependent on autophagy and NF-kB but independent of p53 and it coïncides with DNA damage and a DNA damage response in p53-proficient cells. Our results point out a crosstalk between NF-kB and autophagy controlling YM155-induced death in breast cancer cells and argue for the potential use of YM155 as a genotoxic agent in breast cancer therapy.
