Abstract
Cancer generates a state of immune suppression that contributes to tumor out-growth and the escape phase of the immune editing of nascent tumors. One hallmark of tumor mediated immune suppression is a decreased number of dendritic cells (DC) and an accumulation of immature myeloid cells (myeloid derived suppressor cells, MDSC) that are themselves directly immunosuppressive. Impairment of DC differentiation is mediated by numerous tumor derived factors (VEGF, GM-CSF, IL-6, and M-CSF) that activate Stat3. The mechanism by which Stat3 signaling subsequently inhibits DC differentiation has not been defined. Previous work in our lab has identified protein kinase C βII (PKC βII) as being essential in myeloid progenitor à DC differentiation and that knock down of PKC βII expression blocks DC differentiation. This leads us to hypothesize that tumor derived factor activation of Stat3 may inhibit DC differentiation by down regulating PKC βII expression. To test our hypothesis, we utilized the hematopoietic progenitor-like cell line KG1. In response to the phorbol ester PMA, KG1 differentiates into a DC-like cell (KG1-DC). KG1 cultured in media conditioned by a human (MCF-7) or murine (DA3) mammary cancer cell line had a 48% (DA3) and 51% (MCF-7) reduction in PKC βII protein levels. Additionally, tumor conditioned media treatment significantly decreased PKC βII mRNA transcript levels (38-fold reduction compared to untreated, p<0.01). According to our proposed model, decreased PKC βII expression should inhibit DC differentiation. Consistent with this, KG1 cultured in MCF-7 conditioned media and then differentiated to KG1-DC elicited significantly less allogeneic T cell proliferation (a key measure of DC function) than KG1-DC generated in control media (p<0.01). To test the role of Stat3 activity on PKC βII expression, we have generated a series of clones stably expressing wild type, constitutive active, and dominant negative Stat3 constructs in K562, a second DC progenitor-like cell line. Compared to the parental cell line, PKC βII mRNA transcript levels were significantly reduced (>10-fold) in clones stably expressing the constitutive active Stat3 construct (p<0.1). Clones expressing the dominant negative or wild type Stat3 constructs have PKC βII mRNA transcript levels comparable to or greater than the parental cell line (no difference to 2.5 fold increase). Similarly, PKC βII protein levels in clones stably expressing the constitutive active construct were reduced 75–95% compared to the parental cell line, while PKC βII protein levels in clones expressing the dominant negative construct were increased by 57–110%. We have found that culture of a myeloid progenitor cell line in tumor conditioned media significantly decreases PKC βII mRNA transcript levels and protein levels, suggesting that tumor conditioned media decreases PKC βII gene expression. Decreased PKC βII expression was accompanied by impairment in DC differentiation, as measured by a significant decrease in the ability of KG1-DC to stimulate allogeneic T cell proliferation. Expression of a constitutive active Stat3 construct significantly decreased PKC βII transcript levels in K562, a DC progenitor-like cell line. Taken together, these observations support the hypothesis that tumor derived factors inhibit DC differentiation by down regulation of PKC βII expression via Stat3 hyperactivation.
Exercise-induced changes in β-adrenergic-receptor mRNA level measured by competitive RT-PCR