A Molecular Mechanism of Tumor Mediated Suppression of Dendritic Cell Differentiation

Abstract

1735 The immune system plays a key role in preventing and controlling tumor growth. Cancer frequently induces a state of immune suppression in patients mediated, in part, through inhibition of dendritic cell (DC) differentiation. This results in the accumulation of actively immunosuppressive myeloid derived suppressor cells (MDSCs) and a loss of DCs (critical in the induction of T cell mediated immune responses), thus compromising the ability to initiate anti-tumor immune responses. This is mediated by tumor derived factors (TDFs)(e.g. VEGF) that inhibit DC differentiation by driving STAT3 hyperactivation. The downstream target(s) of this STAT3 signaling that inhibits DC differentiation, however, has not been defined. Previous work in our lab has identified protein kinase C βII (PKC βII) as being essential in myeloid progenitor to DC differentiation and demonstrated that PKC βII inhibition (signaling or expression) prevents DC differentiation. We've also found that PKC βII positively regulates its own expression and that, under certain circumstances, the PKCβ promoter is negatively regulated. These observations lead us to hypothesize that TDF activation of Stat3 inhibits DC differentiation by down regulating PKC βII expression. We've previously shown that culture in tumor conditioned media (TCM) decreased PKC βII protein levels and significantly reduced PKC βII mRNA transcript levels in KG1, a myeloid progenitor-like cell line long used to model DC differentiation. We've also previously seen that decreased PKC βII expression following culture in TCM significantly impaired DC differentiation, compared to cells grown in the absence of tumor conditions; however, the mechanism by which Stat3 signaling down regulated PKC βII expression remained unclear. We now show that culture in TCM reduced PKCβ promoter driven transcription 7-fold, compared to cells grown in normal media (p<0.01). Given the previously described importance of Stat3 hyperactivation in tumor-mediated suppression of DC differentiation, and since PKC βII down regulation appears to occur at the promoter level, we examined the role of Stat3 in regulating PKC βII expression. Culture in TCM rapidly (<5 min.) induced Stat3 phosphorylation, an indication of activation. By chromatin immunoprecipitation, we found that TCM treatment induces direct interaction between Stat3 and the PKCβ promoter, suggesting that Stat3 signaling may act to decrease PKC βII expression. To directly test the role of Stat3 signaling in regulation of PKC βII expression, we generated a series of clones stably expressing wild type (WT) or constitutive active (CA) Stat3 constructs in K562, a second DC progenitor-like cell line. We've previously seen that clones stably expressing the CA-Stat3 construct have decreased PKC βII protein levels and significantly decreased PKC βII mRNA levels, compared to the parental cell line and WT-Stat3 clones. We now show that this decrease in PKC βII expression was dependent on constitutive Stat3 signaling, as pharmacologic Stat3 inhibition restored PKC βII expression to levels seen in the parental cell line. Consistent with the proposed model and our previous work, decreased PKC βII expression in clones expressing CA-Stat3 resulted in significantly inhibited phorbol ester driven DC differentiation (p<0.05)(as measured by allogenic T cell proliferation, a key measure of DC differentiation). Interestingly, we've also found that PKC βII antagonizes Stat3 signaling: myeloid progenitor-like cells (KG1a) overexpressing PKC βII do not exhibit Stat3 activation in response to TCM: PKC βII overexpression or activation led to down regulation of the receptors for G-CSF, IL-6, and VEGF, TDFs demonstrated to inhibit myeloid progenitor to DC differentiation. These findings suggest a novel mechanism by which PKC βII negatively regulates the potential of a cell to respond to (tumor derived) inflammatory cytokines. This work demonstrates that tumor driven Stat3 hyperactivation down regulates PKCβ promoter activity, resulting in decreased PKC βII protein levels. In agreement with our previous work, this decrease in PKC βII expression impairs a cell's potential to undergo DC differentiation. Additionally, this work suggests that PKC βII signaling impairs a cell's potential to signal via Stat3 in response to TDFs, perhaps providing an avenue by which to block or reverse tumor mediated suppression of DC differentiation in cancer. Disclosures: No relevant conflicts of interest to declare.