Tumor-Induced STAT3 Signaling in Myeloid Cells Impairs Dendritic Cell Differentiation by Decreasing Protein Kinase C Beta II Expression
Farren, Matthew Ryan
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The immune system plays a key role in preventing and controlling tumor growth and metastasis, and the ability of nascent tumors to progress is dependent on mechanisms to escape this immune control. One major mechanism is the blockade of dendritic cell (DC) differentiation, resulting in their loss. DCs, central in the induction and regulation of adaptive immune responses, are thought to play an essential role in the immune system's ability to restrain tumor growth and metastasis. Indeed, their abundance in and around tumors is an independent predictor of patient survival. Tumor-mediated inhibition of DC differentiation thus compromises the maintenance and initiation of antitumor immune responses, and additionally may contribute to the accumulation of actively immunosuppressive myeloid derived suppressor cells, further weakening antitumor immunity. Tumor inhibition of DC differentiation is mediated by numerous soluble tumor derived factors (TDFs, e.g. VEGF, IL-6, G-CSF) that accumulate to high levels in the circulation and inhibit DC differentiation by inducing chronic STAT3 activation. Despite its central role in the dysregulation of myelopoiesis in cancer, the molecular mechanisms by which STAT3 blocks DC differentiation remain poorly defined. Our lab previously has identified the serine/threonine kinase protein kinase C beta isoform II (PKC beta II) as being essential in myeloid progenitor-to-DC differentiation and suggested the existence of pathways that negatively regulate its expression. These observations lead us to hypothesize that tumor driven STAT3 activation inhibits DC differentiation by downregulating PKC beta II expression. This work seeks to determine if tumor-induced STAT3 signaling downregulates PKC beta II expression, the mechanism by which it does so, and the effect that this downregulation has on the subsequent ability of a myeloid progenitor cell to undergo DC differentiation. Our analysis of peripheral blood mononuclear cells from patients with advanced breast cancer and splenic myeloid cells from tumor bearing mice found that PKC beta II expression was significantly decreased in the presence of tumor cells. In vitro treatment of myeloid progenitor cells--both primary monocytes and the KG1 cell line model of DC differentiation--with tumor conditioned media (TCM) significantly decreased PKC beta II protein and mRNA abundance, indicating that tumor driven signaling induces cell intrinsic changes in PKC beta II gene expression. Treatment with TCM also significantly impaired the subsequent ability of these cells to undergo DC differentiation. Expression of PKC beta II under the control of an exogenous, viral promoter prevented TCM from downregulating PKC beta II and impairing DC differentiation. This demonstrates that tumor-mediated inhibition of DC differentiation is dependent on PKC beta II downregulation. In the course of these experiments, we were surprised to find that TCM did not induce STAT3 activity in cells overexpressing PKC beta II. We found that these cells had significantly lower expression of the receptors for STAT3 activating TDFs. This suggested that PKC beta II negatively regulates the expression of these receptors, and we tested this by activating PKC beta II with PMA. PKC beta II activation significantly decreased the abundance of these cell surface receptors and likewise decreased their mRNA abundance. This identifies a previously undescribed counter-regulatory mechanism in which PKC beta II activity inhibits tumor-driven STAT3 signaling. TCM rapidly induced significant and sustained (> 3 days) STAT3 activity, the basal activation status of which is negatively correlated with PKC beta II expression. IL-6 was the primary STAT3 activating TDF in our model system, though we believe that this would prove highly context dependent across tumor types. Constitutive STAT3 activation significantly decreased PKC beta II abundance, demonstrating in the absence of other potential signals from TCM that constitutive STAT3 activity downregulates PKC beta II expression. Since STAT3 can repress as well as promote transcription, we examined PKC beta promoter activity and found that it is significantly decreased by culture in TCM. This is mediated by STAT3 binding to a previously undescribed negative regulatory element in the PKC beta promoter and functioning as a transcriptional repressor. Together, these data suggest that a previously unrecognized cross-talk mechanism between the STAT3 and PKC beta II signaling pathways provides the molecular basis for the tumor-induced blockade in the differentiation of myeloid cells into dendritic cells. This work suggests that enhancing PKC beta II activity may be a therapeutic strategy to alleviate cancer-mediated suppression of the immune system.