2 search hits
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Macrophage polarization by apoptotic cancer cells - a RNAi high-throughput screen and validation of interleukin 10 regulation
(2012)
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Stephanie Ley
- Tumor-associated macrophages (TAM) are a major supportive component within neoplasms
and by their plasticity promote all phases of tumor development. Mechanisms of
macrophage (M Phi) attraction and differentiation to a tumor-promoting phenotype, defined
among others by distinct cytokine patterns such as pronounced immunosuppressive
interleukin 10 (IL-10) production, are largely unknown. However, a high apoptosis index
within tumors and strong M Phi infiltration correlate with poor prognosis. Thus, I aimed at
identifying signaling pathways contributing to generation of TAM-like M Phi by using
supernatant of apoptotic cancer cells (ACM) as stimulus.
To distinguish novel factors involved in generating TAM-like M Phi, I used an adenoviral
RNAi-based approach. The primary read-out was production of IL-10. However, mediators
modulating IL-10 were re-validated for their impact on regulation of the cytokines IL-6, IL-8
and IL-12. Following assay development, optimization and down-scaling to a 384-well
format, primary human M Phi were transduced with 8495 constructs of the adenoviral shRNA
SilenceSelect® library of Galapagos BV, followed by activation to a TAM-like phenotype using
ACM. I identified 96 genes involved in IL-10 production in response to ACM and observed a
pronounced cluster of 22 targets regulating IL-10 and IL-6. Principal validation of five targets
of the IL-10/IL-6 cluster was performed using siRNA or pharmacological inhibitors. Among
those, IL-4 receptor-alpha and cannabinoid receptor 2 were confirmed as regulators of IL-10 and
IL-6 secretion.
One protein identified in the screen, the nerve growth factor (NGF) receptor TRKA was
chosen for in-depth validation, based on its involvement in IL-10, IL-6 and IL-12 secretion
from ACM-stimulated human M Phi. TRKA possesses a cardinal role in neuronal development,
but compelling evidence emerges suggesting participation of TRKA in cancer development.
First experiments using pharmacological inhibitors principally confirmed the involvement of
TRKA in IL-10 secretion by ACM-stimulated M Phi and revealed PI3K/AKT and to a lesser
extend MAPK p38 as important signaling molecules downstream of TRKA activation.
Signaling through TRKA required the presence of its ligand NGF, as indicated by NGF
neutralization experiments. NGF was not induced by or present in ACM, but was
constitutively secreted by M Phi. Interestingly, M Phi responded to authentic NGF with neither
AKT and p38 phosphorylation nor IL-10 production. TRKA is well known to be transactivated
by other receptors and in neurons its cellular localization is decisive for its function.
Inhibitors of common transactivation partners did not influence IL-10 production by human
M Phi. Rather, ACM-treatment provoked pronounced translocation of TRKA to the plasma
membrane within 10 minutes as observed by immunofluorescence staining. Consequently, I
was intrigued to clarify mechanisms of TRKA trafficking in response to ACM.
The bioactive lipid sphingosine-1-phosphate (S1P) has been previously identified as
important apoptotic cell-derived mediator involved in TAM-like M Phi polarization. Indeed, I
observed S1P and src kinase involvement in ACM-mediated IL-10 induction. Furthermore,
inhibition of S1P receptor (S1PR) signaling or src kinase activity prevented TRKA
translocation, whereas a TRKA inhibitor or anti-NGF did not block TRKA trafficking to the
plasma membrane in response to ACM. Thus, autocrine secreted NGF activated TRKA to
promote IL-10 secretion, which required previous S1PR/src-dependent translocation of TRKA
to the plasma membrane.
Following the detailed analysis of IL-10 regulation, I was interested whether other TAM
phenotype markers were influenced by ACM and whether their expression was regulated
through TRKA-dependent signaling. Five of six markers were up-regulated on mRNA level by
ACM, and secretion of IL-6, IL-8 and TNF-alpha was triggered. S1PR-signaling was essential for
induction of all but one marker, whereas TRKA signaling was only required for cytokine
secretion. Interestingly, none of the investigated TAM markers was regulated identically to
IL-10, emphasizing a tight and exclusive regulation machinery of this potent
immunosuppressive cytokine.
Finally, I aimed to validate the in vitro findings in human ACM-stimulated M Phi. Therefore, I
isolated murine TAM as well as other major mononuclear phagocyte populations from
primary oncogene-induced breast cancer tissue. Indeed, TRKA-dependent signaling was
required for spontaneous cytokine production selectively by primary murine TAM. Besides
IL-10, the TRKA pathway was decisive for secretion of IL-6, TNF-alpha and monocyte chemotactic
protein-1, indicating its relevance in cancer-associated inflammation.
In summary, my findings highlight a fine-tuned regulatory system of S1P-dependent
TRKA trafficking and autocrine NGF signaling in TAM biology. Both factors, S1P as well as
NGF, might be interesting targets for future cancer therapy.
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Identification of translationally deregulated proteins during inflammation-associated tumorigenesis
(2012)
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Daniela Rübsamen
- The translation of mRNAs into proteins is an elaborate and highly regulated process. Translational regulation primarily takes place at the level of initiation. During initation the eukaryotic initiation factors (eIFs) form a complex that binds to the 5’end of the mRNA to scan for a start codon. Once recognized, the ribosome is recruited to the mRNA and protein synthesis starts. Initiation of translation can basically occur via two distinct mechanisms, i.e. cap-dependent and cap-independent that is mediated via internal ribosome entry sites (IRESs). The former is mediated by a 5’cap structure composed of a 7-methylguanylate which is added to every mRNA during transcription and recruits the initiation complex. IRES-dependent translation involves elements within the 5’untranslated region (UTR) of the mRNA that mostly bind IRES trans-acting factors (ITAFs) which associate either with the initiation complex or with the ribosome itself and consequently allow for internal initiation of translation.
During tumorigenesis the demand for proteins is increased due to rapid cell growth, which consequently requires enhanced translation. Many factors that regulate translation are overexpressed in tumors. Moreover, signaling pathways that trigger translation or further hyperactivated by the surrounding tumor microenvironment. This environment is largely generated by infiltration of immune cells such as macrophages that secrete cytokines and other mediators to promote tumorigenesis. As the effects of inflammatory conditions on the translation of specific targets are only poorly characterized, my study aimed at identifying translationally deregulated targets during inflammation-associated tumorigenesis.
For this purpose, I cocultured MCF7 breast tumor cells with conditioned medium of activated monocyte-derived U937 macrophages (CM). Polysome profiling and microarray analysis identified 42 targets to be regulated at the level of translation. The results were validated by quantitative PCR and one target - early growth response 2 (EGR2) - was chosen for in depth analysis of the mechanism leading to its enhanced translation.
In order to identify upstream signaling molecules causing enhanced EGR2 protein synthesis the cytokine profile of CM was analyzed and the impact of several cytokines on EGR2 translation was examined. Preincubation of CM with neutralizing antibodies revealed that lowering interleukin 6 (IL-6) had only little effect, whereas depletion of IL 1β significantly reduced EGR2 translation. This finding was corroborated by the fact that treatment with recombinant IL-1β enhanced EGR2 translation to virtually the same extend as CM. Further experiments revealed that this effect was mediated via the p38-MAPK signaling cascade.
Interestingly, I observed that the mTOR inhibitor rapamycin, which reduces cap-dependent translation, specifically stimulated EGR2 translation. This result argued for an IRES-dependent mechanism that might account for EGR2 translation. The use of bicistronic reporter assays verified this hypothesis. In line with the above mentioned results, CM, IL-1β and p38-MAPK induced EGR2-IRES activity.
Since IRESs commonly require ITAFs to mediate translation initiation, the binding of proteins to the 5’UTR was analyzed using mass spectrometry. Among others, several previously described ITAFs, such as polypyrimidine tract-binding protein (PTB) and heterogeneous nuclear ribonucleoprotein A1 (hnRNP-A1) were identified to directly bind to the EGR2-5’UTR. Furthermore, overexpression of hnRNP-A1 enhanced EGR2-IRES activity whereas a dominant negative form of hnRNP-A1 significantly decreased it, thus, showing its importance for EGR2 translation.
In summary, my data provide evidence that EGR2 expression can be controlled by IRES-dependent translational regulation, which is responsive to an inflammatory environment. The identified mechanism may not be exclusive for one target but might be representative for gene expression regulation mechanisms during tumorigenesis. This is of special interest for the treatment of cancer patients and development of more specific therapies to reduce tumor outcome.
