Early detection of tumor cells by innate immune cells leads to T(reg) recruitment through CCL22 production by tumor cells.

In breast carcinomas, patient survival seems to be negatively affected by the recruitment of regulatory T cells (T(reg)) within lymphoid aggregates by CCL22. However, the mechanisms underpinning this process, which may be of broader significance in solid tumors, have yet to be described. In this study, we determined how CCL22 production is controlled in tumor cells. In human breast carcinoma cell lines, CCL22 was secreted at low basal levels that were strongly increased in response to inflammatory signals [TNF-α, IFN-γ, and interleukin (IL)-1ß], contrasting with CCL17. Primary breast tumors and CD45(+) infiltrating immune cells appeared to cooperate in driving CCL22 secretion, as shown clearly in cocultures of breast tumor cell lines and peripheral blood mononuclear cells (PBMC) or their supernatants. We determined that monocyte-derived IL-1ß and TNF-α are key players as monocyte depletion or neutralization of these cytokines attenuated secretion of CCL22. However, when purified monocytes were used, exogenous human IFN-γ was also required to generate this response suggesting a role for IFN-γ-producing cells within PBMCs. In this setting, we found that human IFN-γ could be replaced by the addition of (i) IL-2 or K562-activated natural killer (NK) cells or (ii) resting NK cells in the presence of anti-MHC class I antibody. Taken together, our results show a dialogue between NK and tumor cells leading to IFN-γ secretion, which in turn associates with monocyte-derived IL-1ß and TNF-α to drive production of CCL22 by tumor cells and subsequent recruitment of T(reg). As one validation of this conclusion in primary breast tumors, we showed that NK cells and macrophages tend to colocalize within tumors. In summary, our findings suggest that at early times during tumorigenesis, the detection of tumor cells by innate effectors (monocytes and NK cells) imposes a selection for CCL22 secretion that recruits T(reg) to evade this early antitumor immune response.

ADP ribosylation factor like 2 (Arl2) regulates breast tumor aggressivity in immunodeficient mice.

We have previously reported that ADP ribosylation factor like 2 (Arl2), a small GTPase, content influences microtubule dynamics and cell cycle distribution in breast tumor cells, as well as the degree and distribution of phosphorylated P53. Here we show, in two different human breast adenocarcinoma models, that Arl2 content has a major impact on breast tumor cell aggressivity both in vitro and in vivo. Cells with reduced content of Arl2 displayed reduced contact inhibition, increased clonogenic or cluster formation as well as a proliferative advantage over control cells in an in vitro competition assay. These cells also caused larger tumors in SCID mice, a phenotype which was mimicked by the in vivo administration of siRNA directed against Arl2. Cells with increased Arl2 content displayed reduced aggressivity, both in vitro and in vivo, with enhanced necrosis and were also found to contain increased PP2A phosphatase activity. A rt-PCR analysis of fresh human tumor breast samples suggested that low Arl2 expression was associated with larger tumor size and greater risk of lymph node involvement at diagnosis. These data underline the role of Arl2, a small GTPase, as an important regulator of breast tumor cell aggressivity, both in vitro and in vivo.

Regulation of estrogen rapid signaling through arginine methylation by PRMT1.

Evidence is emerging that estrogen receptor alpha (ERalpha) is central to the rapid transduction of estrogen signaling to the downstream kinase cascades; however, the mechanisms underlying this nongenomic function are not fully understood. Here we report a paradigm of ERalpha regulation through arginine methylation by PRMT1, which transiently methylates arginine 260 within the ERalpha DNA-binding domain. This methylation event is required for mediating the extranuclear function of the receptor by triggering its interaction with the p85 subunit of PI3K and Src. Furthermore, we find that the focal adhesion kinase (FAK), a Src substrate involved in the migration process, is also recruited in this complex. Our data indicate that the methylation of ERalpha is a physiological process occurring in the cytoplasm of normal and malignant epithelial breast cells and that ERalpha is hypermethylated in a subset of breast cancers.

Dendritic cell infiltration and prognosis of early stage breast cancer.

PURPOSE: Although dendritic cells (DC) and T cells can infiltrate primary breast carcinoma, it remains unclear whether the immune response influences the clinical outcome. EXPERIMENTAL DESIGN: T lymphocytes and DC infiltration within primary tumors was investigated in 152 patients with invasive nonmetastatic breast cancer. CD1a, CD3, CD68, CD123, CD207/Langerin, and CD208/DC-LAMP expression was assessed with semiquantitative immunohistochemical analysis. Expression of chemokines involved in DC migration (MIP-3a/CCL20, MIP-3b/CCL19, and 6Ckine/CCL21) was also examined. The correlation between these markers and the characteristics of the tumors, as well as relapse-free and overall survival was analyzed. Significant prognostic parameters were then tested in a validation series. RESULTS: Infiltration by immature CD207/Langerin+ DC was found in a third of the cancers and did not correlate with clinicopathological data. Presence of mature CD208/DC-LAMP+ DC (56%) and CD3+ T cells (82%) strongly correlated with lymph node involvement and tumor grade. Among the chemokines analyzed, only the presence of MIP-3b/CCL19 in 57% of the tumors correlated with prolonged overall survival. CD123+ plasmacytoid DC (pDC) infiltrated 13% of the primary tumors. Their presence was strongly associated with shorter overall survival (93% versus 58% at 60 months) and relapse-free survival (90% versus 37% at 60 months) and was found to be an independent prognostic factor for overall survival and relapse-free survival and confirmed in an independent validation series of 103 patients. CONCLUSIONS: Infiltration by pDC of primary localized breast tumor correlates with an adverse outcome, suggesting their contribution in the progression of breast cancer.

Breast carcinoma cells promote the differentiation of CD34+ progenitors towards 2 different subpopulations of dendritic cells with CD1a(high)CD86(-)Langerin- and CD1a(+)CD86(+)Langerin+ phenotypes.

Primary breast carcinoma are frequently infiltrated by dendritic cells (DC). The mechanisms involved in the localization and status of activation of DC within primary breast carcinoma were investigated. CCL20/MIP3alpha, a chemokine involved in immature DC and their precursors attraction, was detected by immunohistochemistry on cryopreserved tissue sections of primary breast tumors and by ELISA and biological assay in metastatic effusion fluids from breast cancer patients but not from other tumors. In vitro, irradiated breast carcinoma cell lines (BCC) as well as their conditioned media promoted CD34+ cell differentiation into CD1a+ Langerhans cells (LC) precursors as early as day 6, while at day 12, 2 different CCR6+ subpopulations of DC with a Langerhans cell (CD1a(+)Langerin(+)CD86+) and an immature DC (CD1a(high)Langerin-CD86(-)HLA-DR(low)CD40(low)) phenotype were observed. This phenomenon was partly driven by a TGFbeta-dependent mechanism since a pan TGFbeta polyclonal antibody completely blocks BCC-induced LC differentiation and partly reduces immature DC development. These DC failed to maturate in response to sCD40L or LPS stimuli and CD1a(high)Langerin(-)CD86- cells have a reduced T-cell stimulatory capacity in MLR experiments. The absolute number of T cells was reduced by 50% in both the CD4+ or CD8+ compartments, these T cells expressing lower levels of the CD25 Ag and producing less IFNgamma. These results show that breast carcinoma cells produce soluble factors, which may attract DC and their precursors in vivo, and promote the differentiation of the latter into LC and immature DC with altered functional capacities. The infiltration of BCC by these altered DC may contribute to the impaired immune response against the tumor.

CD208/dendritic cell-lysosomal associated membrane protein is a marker of normal and transformed type II pneumocytes.

Dendritic cell-lysosomal associated membrane protein (DC-LAMP)/CD208, a member of the lysosomal associated membrane protein (LAMP) family, is specifically expressed by human DCs on activation. However, its mouse counterpart could not be detected in mature DCs. The present study demonstrates that DC-LAMP is constitutively expressed by mouse, sheep, and human type II pneumocytes. Confocal and immunoelectron microscopy showed that mouse DC-LAMP protein co-localizes with lbm180, a specific marker for the limiting membrane of lamellar bodies that contain surfactant protein B, as well as with intracellular MHC class II molecules that accumulate in the same organelles. Expression of DC-LAMP was also occasionally detected at the cell surface of type II pneumocytes. Interestingly, human bronchioloalveolar carcinoma tumor cells, which correspond to transformed type II pneumocytes, express DC-LAMP. Similar observations were made in the Jaagsiekte sheep retrovirus-associated ovine pulmonary adenocarcinoma, a model of human bronchioloalveolar carcinoma. This study establishes that DC-LAMP is constitutively expressed in normal type II pneumocytes. Furthermore, DC-LAMP appears to be a marker of transformed type II pneumocytes as well, an observation that may help the study and the classification of human lung adenocarcinomas.