Tumors evade immune destruction by actively inducing immune tolerance through the recruitment of CD4+CD25+Foxp3+ regulatory T cells (Treg). We have previously described increased prevalence of these cells in pancreatic adenocarcinoma, but it remains unclear what mechanisms are involved in recruiting Treg into the tumor microenvironment. Here, we postulated that chemokines might direct Treg homing to tumor. We show, in both human pancreatic adenocarcinoma and a murine pancreatic tumor model (Pan02), that tumor cells produce increased levels of ligands for the CCR5 chemokine receptor, and, reciprocally, that CD4+ Foxp3+ Treg, compared with CD4+ Foxp3− effector T cells, preferentially express CCR5. When CCR5/CCL5 signaling is disrupted, either by reducing CCL5 production by tumor cells or by systemic administration of a CCR5 inhibitor (TAK-779), Treg migration to tumors is reduced and tumors are smaller than in control mice. Thus, this study demonstrates the importance of Treg in immune evasion by tumors, how blockade of Treg migration may inhibit tumor growth, and, specifically in pancreatic adenocarcinoma, the role of CCR5 in the homing of tumor-associated Treg. Selective targeting of CCR5/CCL5 signaling may represent a novel immunomodulatory strategy for the treatment of cancer.

An important mechanism by which pancreatic cancer avoids anti-tumor immunity is by recruiting regulatory T cells (Treg) to the tumor microenvironment. Recent studies suggest that suppressor Treg and effector Th17 cells share a common lineage and differentiate based on the presence of certain cytokines in the microenvironment. Since IL-6 in the presence of TGF-β has been shown to inhibit Treg development and induce Th17 cells, we hypothesized that altering the tumor cytokine environment could induce Th17 and reverse tumor-associated immune suppression. Pan02 murine pancreatic tumor cells that secrete TGF-β were transduced with the gene encoding IL-6. C57BL/6 mice were injected subcutaneously with wildtype (WT Pan02), empty vector (EV Pan02), or IL-6 transduced Pan02 cells (IL-6 Pan02) to investigate the impact of IL-6 secretion in the tumor microenvironment. Mice bearing IL-6 Pan02 tumors demonstrated significant delay in tumor growth and better overall median survival compared with mice bearing WT or EV Pan02 tumors. Immunohistochemical analysis demonstrated an increase in Th17 cells (CD4+IL-23R+ cells and CD4+IL-17+ cells) in tumors of the IL-6 Pan02 group compared with WT or EV Pan02 tumors. The upregulation of IL-17-secreting CD4+ tumor-infiltrating lymphocytes was substantiated at the cellular level by flow cytometry and ELISPOT assay, and mRNA level for RORγt and IL-23 receptor by RT-PCR. Thus, the addition of IL-6 to the tumor microenvironment skews the balance toward Th17 cells in a murine model of pancreatic cancer. The delayed tumor growth and improved survival suggests that induction of Th17 in the tumor microenvironment produces an anti-tumor effect.

Epithelial-mesenchymal transition (EMT) is an important biologic process that has been implicated in cancer metastasis. Epithelial cell adhesion molecule (EpCAM) is expressed at the basolateral membrane of most normal epithelial cells, but is overexpressed in many epithelial cancers. In our studies of the role of EpCAM in cancer biology, we observed that EpCAM expression is decreased in mesenchymal-like primary cancer specimens in vivo, and following induction of EMT in cancer cell lines in vitro. Extracellular signal-related kinase (ERK) is a key regulator of EMT. We observed that EpCAM expression is decreased with activation of the ERK pathway in primary cancer specimens in vivo and in cancer cell lines in vitro. In experimental models, growth factor stimulation and/or oncogene-induced ERK2 activation suppressed EpCAM expression, whereas genetic or pharmacologic inhibition of the ERK pathway restored EpCAM expression. In detailed studies of the EpCAM promoter region, we observed that ERK2 suppresses EpCAM transcription directly by binding to a consensus ERK2 binding site in the EpCAM promoter, and indirectly through activation of EMT-associated transcription factors SNAI1, SNAI2, TWIST1, and ZEB1, which bind to E-box sites in the EpCAM promoter. Surprisingly, EpCAM appears to modulate ERK activity. Using multiple cell lines, we demonstrated that specific ablation of EpCAM resulted in increased ERK pathway activity, SNAI2 expression, migration and invasion, whereas forced expression of EpCAM resulted in decreased ERK pathway activity, decreased SNAI2 expression, migration and invasion. These observations provide important insights into the regulation of EpCAM expression during EMT, demonstrate an unexpected role for EpCAM in the regulation of ERK, and define a novel double-negative feedback loop between EpCAM and ERK that contributes to the regulation of EMT. These studies have important translational implications as both EpCAM and ERK are currently being targeted in human clinical trials.

p53 is a tumor suppressor gene with well-characterized roles in cell cycle regulation, apoptosis and the maintenance of genome stability. Recent evidence suggests that p53 may also contribute to the regulation of migration and invasion. Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein that is overexpressed in the majority of human epithelial carcinomas, including breast and colorectal carcinomas. We demonstrate by chromatin immunoprecipitation assays that p53 interacts with a candidate p53 binding site within the EpCAM gene. p53-mediated transcriptional repression of EpCAM was confirmed in gain-of-function, and loss-of-function experimental systems. Induction of wildtype p53 was associated with a significant dose-dependent decrease in EpCAM expression; conversely, specific ablation of p53 was associated with a significant increase in EpCAM expression. At the functional level, specific ablation of p53 expression is associated with increased breast cancer invasion, and this effect is abrogated by concomitant specific ablation of EpCAM expression. Taken together, these biochemical and functional data are the first demonstration that (1) wildtype p53 protein binds to a response element within the EpCAM gene and negatively regulates EpCAM expression, and (2) transcriptional repression of EpCAM contributes to p53 control of breast cancer invasion.

IntroductionEpCAM is a cell-surface glycoprotein that is overexpressed in the majority of epithelial carcinomas. However, the functional role of EpCAM in regulating cancer invasion remains controversial, and the mechanism(s) underlying EpCAM-mediated regulation of breast cancer invasion remain to be defined.MethodsEpCAM expression was manipulated in breast cancer cell lines using RNA interference and cDNA expression constructs. Recombinant EpCAM was used to rescue EpCAM signaling following specific ablation of EpCAM. Protein and gene expression, invasion, transcription factor activity, and protein phosphorylation were measured using standard molecular biology techniques.ResultsIn loss-of-function, and gain-of-function experiments we demonstrate that EpCAM expression is associated with increased breast cancer invasion in vitro and in vivo. We demonstrate further that specific ablation of EpCAM expression is associated with decreased activator protein-1 (AP-1) transcription factor activity. Phosphoprotein analyses confirm that specific ablation of EpCAM is associated with decreased phosphorylation of the AP-1 subunit c-Jun. Recombinant soluble extracellular EpCAM (rEpCAM) is able to rescue invasion, AP-1 transcription factor activity, and c-Jun phosphorylation in a dose-dependent fashion. Pharmacologic inhibitors, and constitutively active constructs of the c-Jun N-terminal kinase (JNK) signal transduction pathway, suggest that the impact of EpCAM expression on AP-1 transcription factor activity is mediated through the JNK pathway. In functional rescue experiments, forced expression of c-Jun rescues invasion in breast cancer cells following specific ablation of EpCAM.ConclusionsThese data demonstrate for the first time that EpCAM expression can influence the JNK/AP-1 signal transduction pathway, and suggest that modulation of AP-1 transcription factor activity contributes to EpCAM-dependent breast cancer invasion. These data have important implications for the design and application of molecular therapies targeting EpCAM.