Apoptoseresistenz und Vermeidung von anti-Tumor-Immunität –zwei verwandte (?) Überlebensstrategien maligner Gliomzellen

Our improved understanding of cancer biology has helped to establish criteria that appear to be prerequisites for tumour formation by transformed cells. Cancer cells must (i) be able to grow autonomously, (ii) develop insensitivity to negative growth regulation, (iii) display unlimited replicative potential, (iv) develop the capacity for angiogenesis, (v) develop competence for invasive growth and metastasis and (vi) evade intrinsic apoptotic signals. A further emerging precondition for the malignant phenotype in vivo is the capacity of a malignant cell to evade the extrinsic tumour suppressor functions of the immune system. Glioma cells display all of these “hallmarks of cancer”. Their intrinsic resistance to apoptosis and their immunosuppressive properties (chapter 1) have been analyzed in our laboratory and were also the main topics explored in my Ph.D. work. p53 is a potent activator of the intrinsic apoptotic pathway. It represents an ideal target for anti-cancer drug design, since it is mutated in more than half of human tumours. Most of the remaining tumours, although carrying wild-type p53, have defects in the p53-mediated apoptotic pathway. Unlike most other tumour suppressor genes, mutant p53 is overexpressed in tumour cells. Therefore it can function as a tumour antigen. However, the approach that was investigated here (chapter 2) aimed at restoring a tumour-suppressor function in mutant p53 variants. CP-31398 is a prototype small molecule that stabilizes the active conformation of p53 and promotes p53 activity in cancer cell lines with mutant or wild-type p53. CP-31398 was found to induce p53-like activity in all of 11 glioma cell lines harbouring wild-type or mutant p53, but not in p53 null LN-308 cells. However, upon prolonged exposure to CP-31398, all glioma cell lines, irrespective of their p53 status, undergo caspase-independent cell death displaying some characteristics of apoptosis. Posttranscriptional repression of p53 by an intracellularly transcribed siRNA as well as analysis of p53 transgenes in p53-deleted glioma cells allowed to delineate two pathways of CP-31398-induced cell death: an early, p53-dependent pathway that requires new p53 protein synthesis and a late, p53-independent pathway characterized by calcium release and epiphenomenal free radical formation. These observations point out some of the liabilities of CP-31398 as a prototype p53-based therapeutic and define a rationale for the further refinement of small molecules which specifically target the p53 pathway, but lack the p53-independent effects. At the current stage of development, CP-31398 has promising effects on mutant and wild-type p53 in cancer cell lines, but its strong p53-independent side effects are likely to prevent its use in the clinic. Surprisingly, posttranscriptional repression of p53 by an intracellularly transcribed siRNA in a p53 mt cell line resulted in the induction of a low, but detectable p53-like reporter gene activity. This observation prompted us to investigate the specificity of Stratagene´s p53 PathDetect reporter plasmid (chapter 3), a widely used luciferase-coupled reporter gene for the detection of the transcriptional activity of p53. Both p63 and p73 were found to activate the promoter sequence contained in the p53-luc vector. This shows that data obtained using this system (or any similar one) should be interpreted with caution because they may not actually reflect p53 activity, but rather provide an integrated signal derived from different members of the p53 family. However, this does not seriously challenge the reporter gene data obtained with CP-31398, since a net effect of CP-31398 on p53, p63 and p73 is a physiologically perfectly sensible measure of CP-31398-dependent p53-related activity. In fact, treatment of glioma cells with CP-31398 also increases the cellular level of p73, while p63 is absent from most glioma cell lines (data not shown). Further, the potency of CP-31398 as an inducer of apoptosis may even be enhanced by a cooperative effect on further members of the p53 protein family. Whereas in glioma cells CP-31398 activates an intrinsic, caspase-independent cell death pathway, the extrinsic or death receptor mediated apoptotic pathway may also potentially be exploited for an anti-cancer therapy, since the distribution of death receptors found on tumour cells apparently differs from that found on non-transformed cells. Further, the signalling originating from these receptors may be different in tumour cells. This has been described for CD40, one of several members of the TNFR family that lack a death domain and yet can induce apoptosis. Physiologically, CD40 transduces antiapoptotic signalling in B and T cells whereas CD40-mediated apoptosis appears to be restricted to transformed cells. Therefore adverse effects like the massive hepatotoxicity of CD95/FasL are unlikely to occur with CD40. Instead, ligation of CD40 can induce antitumour immunity in vivo in mice. CD40 is expression was detected in all of 12 human glioma cell lines (chapter 4), even though in most cell lines the expression seems to be mainly intracellular. In contrast to various non-glial carcinoma cell lines, CD40-expressing glioma cells resist cytotoxic effects of CD40L. Resistance to CD40L-induced cell death has been described as the result of CD40 downregulation during long-term culture. CD40 gene transfer into LN-18 glioma cells induces (or restores?) susceptibility to cell death by soluble CD40L. Similar to CD95L- or Apo2L/TRAIL-induced apoptosis, CD40-dependent cell death is potentiated by CHX and involves receptor clustering and caspase 8 and 3 processing. Surprisingly, CD40-transfected LN-18 cells acquire resistance to CD95L, resulting from the down-regulation of CD95 expression via a posttranslational, proteasome inhibitor-sensitive mechanism. In contrast, subtoxic concentrations of CD40L strongly sensitize these cells for TNF-alpha-induced apoptosis via a mechanism involving intracellular crosstalk between CD40 and TNF-R1 (p55). This suggests complex patterns of modulation of death receptor-mediated glioma cell apoptosis by CD40/CD40L interactions. Further, bispecific CD40xCD95, but not CD20xCD95, antibodies kill glioma cells, disclosing the property of endogenous CD40 to facilitate death signalling. However, the data obtained on CD40 in glioma cells do not strongly support a therapeutical application of CD40L as an inducer of apoptosis. On the other hand, CD40L had no stimulatory or antiapoptotic effects on glioma cells and could therefore be used as an adjuvant in a potential anti-glioma immunotherapy. Another TNFR-related cell surface receptor implicated in T, B and NK cell activation as well as in apoptosis is CD27. The ligand for CD27, CD70 was identified as a radio-inducible gene in U87MG glioma cells (chapter 5). A screening of a panel of human glioma cell lines revealed that 11 of 12 cell lines expressed CD70 mRNA and protein. CD70 protein was also detected by immunocytochemistry in 5 of 12 glioblastomas and 3 of 4 anaplastic astrocytomas in vivo. CD27 expression was not detected in any glioma cell line, and there was no evidence for autocrine or backward signalling of the CD70 system in human glioma cells. Unexpectedly, CD70 expressed on glioma cells did not increase the immunogenicity of glioma cells in vitro. In contrast, CD70-positive glioma cells induced apoptosis in PBMC in a CD70-dependent manner. Neutralization of CD70 expressed on glioma cells prevented apoptosis and enhanced the release of TNF-alpha in cocultures of glioma cells and PBMC. The effects of CD70-expressing glioma cells on PBMC were mimicked by agonistic CD27 antibodies. Conversely, the shedding of CD27 by PBMC was identified as a possible escape mechanism from glioma cell-induced CD70-dependent apoptosis. Thus, induction of B cell and T cell apoptosis via interactions of CD70 expressed on glioma cells and CD27 expressed on B and T cells may be a novel way for the immune escape of malignant gliomas. Our in vitro results are in contrast to in vivo experiments describing CD27-mediated tumour rejection. Further, a study using CD27 knockouts supports a role for CD27 in costimulation. However, chronic stimulation via CD70 as occurs in CD70 transgenic mice results in B cell depletion and lethal T cell immunodeficiency. Further, the immune system of glioma patients does not reject CD70-expressing tumours in vivo. Instead, the high proportion of CD70-positive gliomas found in this and a related study suggests that there may rather be a selection pressure operating in favour of CD70-expressing glioma cells. These apparently contradictory data suggest that CD27 may be functioning in vivo together with other receptors that determine the outcome of CD27 stimulation. Thus CD27 may signal either activation or apoptosis, depending on the context. Given the capability of glioma cells to paralyse anti-tumour immune reactions, it is well conceivable that the stimulatory pathways originating from CD27 are inhibited in glioma patients whereas the pathway leading to T cell apoptosis may be enforced. One newly discovered mechanism by which glioma cells apparently inhibit anti-tumour T cell responses is expression and secretion of HLA-G molecules (chapter 6). HLA-G is a nonclassical MHC molecule with highly limited tissue distribution that has been implicated in foetal semi-allograft tolerance during pregnancy. To delineate the potential role of HLA-G in glioblastoma immunobiology, expression patterns and functional relevance of this MHC class Ib molecule were investigated in glioma cell lines and brain tissue. HLA-G protein was detected in 4 of 12 cell lines in the absence, and in 8 out of 12 cell lines in the presence of IFN-gamma. Immunohistochemical analysis of human brain tumours revealed expression of HLA-G in 4 of 5 tissue samples. Functional studies showed that expression of membrane-bound HLA-G1 and soluble HLA-G5 inhibited alloreactive and antigen-specific immune responses. However, HLA-G dependent T cell apoptosis was not detected. Gene transfer of HLA-G1 or HLA-G5 into HLA-G-negative glioma cells rendered these cells highly resistant to direct alloreactive lysis, inhibited the alloproliferative response and prevented efficient priming of cytotoxic T cells. The inhibitory effects of HLA-G were directed against CD8+ and CD4+ T cells, but appeared to be NK cell-independent. Interestingly, few HLA-G-positive cells within a population of HLA-G-negative tumour cells exerted significant immune inhibitory effects. These data suggest that aberrant expression of HLA-G may contribute to immune escape in human glioblastoma. A long-established determinant of glioblastoma-induced immunosuppression is the cytokine TGF-beta. However, TGF-beta does not only compromise anti-tumour immune responses and induce T cell apoptosis, but also stimulates angiogenesis and invasion. Therefore, TGF-beta has become a major target for the experimental treatment of human malignant gliomas. The effects of TGF-beta on NKG2D-mediated anti-tumour immunity were investigated (chapter 8). NKG2D is an activating immunoreceptor expressed by NK, CD8+ alpha beta and gamma delta T cells. Human NKG2DL comprise MHC class I-chain related molecules A (MICA) and MICB and UL16-binding proteins (ULBP) 1, 2, 3, and 4. These ligands are expressed by infected and transformed cells and transmit danger signals to immune cells, leading to the lysis of NKG2DL-expressing cells. An effect of TGF-beta on the expression of the activating immunoreceptor NKG2D in CD8+ T and NK cells has been shown by us and others in vitro. Accordingly, a downregulation of NKG2D is also observed in glioblastoma patients in vivo. Moreover, TGF-beta inhibits the transcription of the NKG2D ligands MICA, ULBP2 and ULBP4 and reduces their protein level at the cell surface. In contrast, MICB, ULBP1 and ULBP3 are unaffected by TGF-beta. Further, TGF-beta promotes the release of matrix metalloproteinases (MMP) by glioma cells which in turn mediate the shedding of MICA from the surface of glioma cells. siRNA-mediated “knock-down“ of TGF-beta synthesis in LN-229 glioma cells suppresses MMP expression and strongly enhances the immunogenicity of glioma cells by enhancing MICA, ULBP2 and ULBP4 expression and by preventing NKG2D down-regulation on immune cells treated with glioma cell SN. Further, the TGF-beta knock-down leads to a decreased migratory and invasive tumour phenotype. Altogether, the knock-down of TGF-beta results in a glioma cell phenotype which is less motile, more sensitive to immune cell lysis, and non-tumorigenic in nude mice. These data define downregulation of NKG2D expression on immune effector cells and of NKG2DL expression on glioma cells as a further mechanism by which TGF-beta may promote immune escape. This confirms the importance of TGF-beta as a principle inhibitor of anti-glioma immune responses. Strategies to counteract the immunosuppressive activities of TGF-beta may be crucial to improve the current cancer immunotherapeutic approaches. In this context, a novel TGF-beta RI kinase inhibitor, SD-208, was characterized for its effects on the growth and immunogenicity of murine SMA-560 and human LN-308 glioma cells in vitro and the growth of, and immune response to, intracranial SMA-560 gliomas in syngeneic VM/Dk mice in vivo (chapter 7). SD-208 blocks autocrine and paracrine TGF-beta signalling in glioma cells as detected by phosphorylation of Smad2 or TGF-beta reporter assays and strongly inhibits constitutive and TGF-beta-evoked migration and invasion. PBL or purified T cells, cocultured with TGF-beta-releasing LN-308 glioma cells in the presence of SD-208, exhibit enhanced lytic activity against LN-308 targets. The release of IFN-gamma and TNF-alpha by these immune effector cells is enhanced by SD-208 whereas the release of IL-10 is reduced. SD-208 restores the lytic activity of polyclonal NK cells against glioma cells in the presence of recombinant TGF-beta or TGF-beta-containing glioma cell SN. The oral bioavailability of SD-208 was verified by demonstrating the inhibition of TGF-beta-induced Smad phosphorylation in spleen and brain. Systemic SD-208 treatment initiated three days after the implantation of SMA-560 cells into the brains of syngeneic VM/Dk mice prolongs their median survival from 18.6 to 25.1 days. Importantly, SD-208 could be administered together with adjuvant chemotherapy or an immunotherapeutic vaccine. Such a combined treatment might further improve the effect achieved by SD-208 alone. Thus, TGF-beta RI kinase inhibitors such as SD-208 are promising novel agents for the treatment of human malignant glioma and other conditions associated with pathological TGF-beta activity. In my Ph.D. research I have thus explored several novel approaches aiming at the selective induction of apoptosis in glioma cells and at an improved understanding of the immune-paralysing capacities of glioma cells. While the connections between these various aspects of glioma biology may not be apparent at first sight, there are good reasons for taking a combined look at apoptosis and tumour immunology: Tumour immunosurveillance is mediated by immune effector cells that selectively induce apoptosis in cancer cells. Activated T and NK attack their targets by expression death ligands and by secretion cytotoxic granules and thus seek to activate both the death receptor and the intracellular apoptotic pathway. An apoptosis-resistant target cell is therefore less susceptible to immune-mediated lysis. Sensitization of tumour cells for apoptotic stimuli could therefore enhance the efficacy of ongoing anti-tumour immune responses. Further, the tumour may strike back and induce apoptosis in tumour infiltrating lymphocytes by various mechanisms, including the expression of CD95/FasL, HLA-G, TGF-beta and, under specific circumstances, CD70 (see section 5). Prevention of tumour cell-induced apoptosis of immune effector cells could also support endogenous anti-tumour responses. Finally, therapeutic induction of apoptosis in tumour cells is thought to provide a source of antigens that can be taken up and presented by professional APC, resulting in an anti-tumour immune response. This, of course, implies that the low rates of spontaneously occurring apoptosis in vivo are simply insufficient to trigger an anti-glioma immune response. Further, the mounting of a productive anti-tumour immune response may be prevented by immune-inhibitory signals sent out by the tumour itself. Therefore, an apoptosis-based reduction of large tumour masses may require the simultaneous relief of tumour-dependent immunosuppression in order to be followed by a productive immune response that can finally clear residual glioma cells dispersed in the brain. Whether the emerging concepts outlined in this Ph.D. thesis have the potential to improve current glioma treatments will, of course, have to be explored in further models. Thus, the present work finally confirms George Bernard Shaw’s notion: “Science... never solves a problem without creating ten more.”Gliomzellen zeichnen sich durch Resistenz gegenüber apoptotischen Stimuli aus. Die Überwindung dieser intrinsischen Apoptose-Resistenz ist daher ein wichtiges Ziel der Gliomforschung. Therapeutische Induktion von Apoptose in Tumorzellen führt in vivo zur Bereitstellung von antigenem Tumorzellmaterial. Dieses kann von dendritischen Zellen aufgenommen und präsentiert werden, sodass eine anti-Tumor-Immunantwort ermöglicht wird. Allerdings verfügen Gliomzellen über eine Reihe immuninhibitorisch wirksamer Mechanismen, die eine Tumorabstoßung verhindern. Zudem stellt die Apoptose-Resistenz von Gliomzellen auch für einen immuntherapeutischen Ansatz ein Hindernis dar, da immun-vermittelte anti-Tumor-Effekte ebenfalls auf der (durch NK und T Zellen vermittelten) Induktion von Apoptose in Krebszellen beruhen. Darüber hinaus können Immunzellen bei Kontakt mit Gliomzellen ebenfalls apoptotisch werden. Daher befasst sich diese Arbeit mit den zellbiologischen Prinzipien des programmierten Zelltods (Apoptose) in Gliomzellen sowie mit den wechselseitigen Beziehungen zwischen Immuneffektorzellen und Gliomzellen, ebenfalls unter besonderer Berücksichtigung der Apoptose. Ein interessantes Agens zur Induktion von Apoptose in Tumorzellen ist das experimentelle Pharmakon CP-31398, das von der Firma Pfizer in den USA mit dem Ziel der Wiederherstellung funktioneller Aktivität in mutanten p53-Proteinen entwickelt wurde. Zur Charakterisierung dieser Substanz wurde unter Zuhilfenahme verschiedene Gentransfer-Techniken ein p53-abhängiger und ein p53-unabhängiger Zelltodweg charakterisiert, die beide durch CP-31398 aktiviert werden. Somit erwies sich CP-31398 als interessante, für den klinischen Gebrauch auf Grund unspezifischer Effekte jedoch nicht hinreichend weit entwickelte Substanz. Die erzielten Ergebnisse könnten aber für die Entwicklung verbesserter p53-modulierender anti-Tumor-Agenzien von Bedeutung sein. Im Rahmen dieser Untersuchungen stellte sich heraus, dass klassische Reporter-Assays für transkriptionelle p53-Aktivität nicht ausschließlich die Aktivität von p53 reflektieren, sondern ein umfassendes Bild der Gesamt-Aktivität von p53, p63, p73 und möglicherweise weiterer, noch nicht bekannter Mitglieder dieser Proteinfamilie liefern. Aufgrund dieses Befundes müssen zahlreiche bisher mit derartigen Reporter-Assays publizierte Daten neu interpretiert werden. In Ergänzung zu CP-31398, das auf die Aktivierung des intrinsischen apoptotischen Mechanismus abzielt (und in Gliomzellen Caspasen-unabhängigen Zelltod induziert), wurde auch der extrinsische oder rezeptor-vermittelte Apoptosemechanismus untersucht. Die bekannten Todesrezeptoren gehören zur Familie der Tumor-Nekrose-Faktor (TNF)-Rezeptoren, die auch bei Immunreaktionen eine wichtige Rolle spielen. Da sich das Expressionsmuster dieser Rezeptoren auf Tumorzellen von demjenigen auf nicht-transformierten Zellen unterscheidet, bieten Todesrezeptoren potentielle therapeutische Angriffspunkte. Systemische Aktivierung des CD95/Fas-Rezeptors führt aber in Mäusen zu Leberversagen und auch die gegenwärtig verfügbaren Präparationen von Apo2L/TRAIL sind klinisch nicht einsetzbar oder ineffektiv. CD40L vermag nach dem bisherigen Kenntnisstand nur in transformierten Zellen Apoptose zu induzieren, was auf eine veränderte Signaltransduktion in Tumorzellen hindeutet. Zudem ist CD40L bereits in immuntherapeutischen Studien klinisch erprobt. Daher wurde im Kontext dieser Arbeit erstmals die Expression und funktionelle Bedeutung von CD40 auf Gliomen untersucht. Dabei wurde CD40-Expression auf allen 12 untersuchten Gliomzelllinien sowie in vivo nachgewiesen. Dennoch wurden keine zytotoxischen Effekte von CD40L auf Gliomzelllinien beobachtet. Da bei Tumoren eine Herunterregulation der CD40-Expression im Verlauf von Langzeit-Kulturen beschrieben ist, wurde eine CD40-überexprimierende Gliomzelllinie generiert. In dieser induziert CD40L Caspasen-abhängigen Zelltod, der durch Hemmung der Proteinbiosynthese potenziert wird. Gleichzeitig kommt es zu einem verstärkten proteasomalen Abbau von CD95/Fas, wodurch die Zellen ihre Sensitivität gegenüber CD95/Fas-vermittelter Apoptose weitgehend verlieren. TNF-alpha-abhängige Apoptose wird hingegen durch subtoxische Konzentrationen von CD40L verstärkt, was durch eine neu entdeckte intrazelluläre Wechselwirkung zwischen CD40 und TNF R1 (p55) erklärt werden kann. Schließlich wurde gezeigt, dass die endogene CD40-Expression von Gliomzellen dazu benützt werden kann, Apoptose mittels eines bispezifischen CD40xCD95 Antikörpers zu induzieren. Somit wurden in diesem Teil der Arbeit komplexe Wechselwirkungen zwischen CD40 und verschiedenen anderen

High-Level Expression of Wild-Type p53 in Melanoma Cells is Frequently Associated with Inactivity in p53 Reporter Gene Assays

Background: Inactivation of the p53 pathway that controls cell cycle progression, apoptosis and senescence, has been proposed to occur in virtually all human tumors and p53 is the protein most frequently mutated in human cancer. However, the mutational status of p53 in melanoma is still controversial; to clarify this notion we analysed the largest series of melanoma samples reported to date. Methodology/Principal Findings: Immunohistochemical analysis of more than 180 melanoma specimens demonstrated that high levels of p53 are expressed in the vast majority of cases. Subsequent sequencing of the p53 exons 5–8, however, revealed only in one case the presence of a mutation. Nevertheless, by means of two different p53 reporter constructs we demonstrate transcriptional inactivity of wild type p53 in 6 out of 10 melanoma cell lines; the 4 other p53 wild type melanoma cell lines exhibit p53 reporter gene activity, which can be blocked by shRNA knock down of p53. Conclusions/Significance: In melanomas expressing high levels of wild type p53 this tumor suppressor is frequently inactivated at transcriptional level

A Novel Mechanism of Soluble HLA-G Mediated Immune Modulation: Downregulation of T Cell Chemokine Receptor Expression and Impairment of Chemotaxis

BACKGROUND: In recent years, many immunoregulatory functions have been ascribed to soluble HLA-G (sHLA-G). Since chemotaxis is crucial for an efficient immune response, we have investigated for the first time the effects of sHLA-G on chemokine receptor expression and function in different human T cell populations. METHODOLOGY/PRINCIPAL FINDINGS: T cell populations isolated from peripheral blood were stimulated in the presence or absence of sHLA-G. Chemokine receptors expression was evaluated by flow cytometry. sHLA-G downregulated expression of i) CCR2, CXCR3 and CXCR5 in CD4(+) T cells, ii) CXCR3 in CD8(+) T cells, iii) CXCR3 in Th1 clones iv) CXCR3 in TCR Vdelta2gamma9 T cells, and upregulated CXCR4 expression in TCR Vdelta2gamma9 T cells. sHLA-G inhibited in vitro chemotaxis of i) CD4(+) T cells towards CCL2, CCL8, CXCL10 and CXCL11, ii) CD8(+) T cells towards CXCL10 and CXCL11, iii) Th1 clones towards CXCL10, and iv) TCR Vdelta2gamma9 T cells towards CXCL10 and CXCL11. Downregulation of CXCR3 expression on CD4+ T cells by sHLA-G was partially reverted by adding a blocking antibody against ILT2/CD85j, a receptor for sHLA-G, suggesting that sHLA-G downregulated chemokine receptor expression mainly through the interaction with ILT2/CD85j. Follicular helper T cells (T(FH)) were isolated from human tonsils and stimulated as described above. sHLA-G impaired CXCR5 expression in T(FH) and chemotaxis of the latter cells towards CXCL13. Moreover, sHLA-G expression was detected in tonsils by immunohistochemistry, suggesting a role of sHLA-G in local control of T(FH) cell chemotaxis. Intracellular pathways were investigated by Western Blot analysis on total extracts from CD4+ T cells. Phosphorylation of Stat5, p70 s6k, beta-arrestin and SHP2 was modulated by sHLA-G treatment. CONCLUSIONS/SIGNIFICANCE: Our data demonstrated that sHLA-G impairs expression and functionality of different chemokine receptors in T cells. These findings delineate a novel mechanism whereby sHLA-G modulates T cell recruitment in physiological and pathological conditions

Cancer Stem Cell Immunology: Key to Understanding Tumorigenesis and Tumor Immune Escape?

Cancer stem cell (CSC) biology and tumor immunology have shaped our understanding of tumorigenesis. However, we still do not fully understand why tumors can be contained but not eliminated by the immune system and whether rare CSCs are required for tumor propagation. Long latency or recurrence periods have been described for most tumors. Conceptually, this requires a subset of malignant cells which is capable of initiating tumors, but is neither eliminated by immune cells nor able to grow straight into overt tumors. These criteria would be fulfilled by CSCs. Stem cells are pluripotent, immune-privileged, and long-living, but depend on specialized niches. Thus, latent tumors may be maintained by a niche-constrained reservoir of long-living CSCs that are exempt from immunosurveillance while niche-independent and more immunogenic daughter cells are constantly eliminated. The small subpopulation of CSCs is often held responsible for tumor initiation, metastasis, and recurrence. Experimentally, this hypothesis was supported by the observation that only this subset can propagate tumors in non-obese diabetic/scid mice, which lack T and B cells. Yet, the concept was challenged when an unexpectedly large proportion of melanoma cells were found to be capable of seeding complex tumors in mice which further lack NK cells. Moreover, the link between stem cell-like properties and tumorigenicity was not sustained in these highly immunodeficient animals. In humans, however, tumor-propagating cells must also escape from immune-mediated destruction. The ability to persist and to initiate neoplastic growth in the presence of immunosurveillance - which would be lost in a maximally immunodeficient animal model - could hence be a decisive criterion for CSCs. Consequently, integrating scientific insight from stem cell biology and tumor immunology to build a new concept of "CSC immunology" may help to reconcile the outlined contradictions and to improve our understanding of tumorigenesis

Tumor Suppressor p53 Alters Host Cell Metabolism to Limit Chlamydia trachomatis Infection

Obligate intracellular bacteria depend entirely on nutrients from the host cell for their reproduction. Here, we show that obligate intracellular Chlamydia downregulate the central tumor suppressor p53 in human cells. This reduction of p53 levels is mediated by the PI3K-Akt signaling pathway, activation of HDM2, and subsequent proteasomal degradation of p53. The stabilization of p53 in human cells severely impaired chlamydial development and caused the loss of infectious particle formation. DNA-damage-induced p53 interfered with chlamydial development through downregulation of the pentose phosphate pathway (PPP). Increased expression of the PPP key enzyme glucose-6-phosphate dehydrogenase rescued the inhibition of chlamydial growth induced by DNA damage or stabilized p53. Thus, downregulation of p53 is a key event in the chlamydial life cycle that reprograms the host cell to create a metabolic environment supportive of chlamydial growth

Growth/Differentiation Factor-15 (GDF-15): From Biomarker to Novel Targetable Immune Checkpoint

International audienceGrowth/differentiation factor-15 (GDF-15), also named macrophage inhibitory cytokine-1, is a divergent member of the transforming growth factor β superfamily. While physiological expression is barely detectable in most somatic tissues in humans, GDF-15 is abundant in placenta. Elsewhere, GDF-15 is often induced under stress conditions, seemingly to maintain cell and tissue homeostasis; however, a moderate increase in GDF-15 blood levels is observed with age. Highly elevated GDF-15 levels are mostly linked to pathological conditions including inflammation, myocardial ischemia, and notably cancer. GDF-15 has thus been widely explored as a biomarker for disease prognosis. Mechanistically, induction of anorexia via the brainstem-restricted GDF-15 receptor GFRAL (glial cell-derived neurotrophic factor [GDNF] family receptor α-like) is well-documented. GDF-15 and GFRAL have thus become attractive targets for metabolic intervention. Still, several GDF-15 mediated effects (including its physiological role in pregnancy) are difficult to explain via the described pathway. Hence, there is a clear need to better understand non-metabolic effects of GDF-15. With particular emphasis on its immunomodulatory potential this review discusses the roles of GDF-15 in pregnancy and in pathological conditions including myocardial infarction, autoimmune disease, and specifically cancer. Importantly, the strong predictive value of GDF-15 as biomarker may plausibly be linked to its immune-regulatory function. The described associations and mechanistic data support the hypothesis that GDF-15 acts as immune checkpoint and is thus an emerging target for cancer immunotherapy

Targeting breast cancer stem cells with HER2-specific antibodies and natural killer cells

Breast cancer is the most common cancer among women worldwide. Every year, nearly 1.4 million new cases of breast cancer are diagnosed, and about 450.000 women die of the disease. Approximately 15-25% of breast cancer cases exhibit increased quantities of the trans-membrane receptor tyrosine kinase human epidermal growth factor receptor 2 (HER2) on the tumor cell surface. Previous studies showed that blockade of this HER2 proto-oncogene with the antibody trastuzumab substantially improved the overall survival of patients with this aggressive type of breast cancer. Recruitment of natural killer (NK) cells and subsequent induction of antibody-dependent cell-mediated cytotoxicity (ADCC) contributed to this beneficial effect. We hypothesized that antibody binding to HER2-positive breast cancer cells and thus ADCC might be further improved by synergistically applying two different HER2-specific antibodies, trastuzumab and pertuzumab. We found that tumor cell killing via ADCC was increased when the combination of trastuzumab, pertuzumab, and NK cells was applied to HER2-positive breast cancer cells, as compared to the extent of ADCC induced by a single antibody. Furthermore, a subset of $CD44^{high}CD24^{low}HER2^{low}$ cells, which possessed characteristics of cancer stem cells, could be targeted more efficiently by the combination of two HER2-specific antibodies compared to the efficiency of one antibody. These in vitro results demonstrated the immunotherapeutic benefit achieved by the combined application of trastuzumab and pertuzumab. These findings are consistent with the positive results of the clinical studies, CLEOPATRA and NEOSPHERE, conducted with patients that had HER2-positive breast cancer. Compared to a single antibody treatment, the combined application of trastuzumab and pertuzumab showed a stronger ADCC effect and improved the targeting of breast cancer stem cells

Anti-CD39 and anti-CD73 antibodies A1 and 7G2 improve targeted therapy in ovarian cancer by blocking adenosine-dependent immune evasion

The ectonucleotidases CD39 and CD73 degrade ATP to adenosine which inhibits immune responses via the $A_{2A}$ adenosine receptor (ADORA2A) on T and NK cells. The current study investigates the potential therapeutic use of the specific anti CD39- and anti CD73-antibodies A1 (CD39) and 7G2 (CD73) as these two ectonucleotidases are overexpressed in ovarian cancer (OvCA). As expected, NK cell cytotoxicity against the human ovarian cancer cell lines OAW-42 or SK-OV-3 was significantly increased in the presence of A1 or 7G2 antibody. While this might partly be due to antibody-dependent cell-mediated cytotoxicity, a luciferase-dependent assay for quantifying biologically active adenosine further showed that A1 and 7G2 can inhibit CD39 and CD73-dependent adenosine-generation. In turn, the reduction in adenosine levels achieved by addition of A1 and 7G2 to OAW-42 or SK-OV-3 cells was found to de-inhibit the proliferation of $CD4^+$ T cells in coculture with OvCA cells. Likewise, blocking of CD39 and CD73 on OvCA cells via A1 and 7G2 led to an increased cytotoxicity of alloreactive primed T cells. Thus, antibodies like A1 and 7G2 could improve targeted therapy in ovarian cancer not only by specifically labeling overexpressed antigens but also by blocking adenosine-dependent immune evasion in this immunogenic malignancy