The dual-function chemokine receptor CCR2 drives migration and chemokine scavenging through distinct mechanisms.

C-C chemokine receptor 2 (CCR2) is a dual-function receptor. Similar to other G protein-coupled chemokine receptors, it promotes monocyte infiltration into tissues in response to the chemokine CCL2, and, like atypical chemokine receptors (ACKRs), it scavenges chemokine from the extracellular environment. CCR2 therefore mediates CCL2-dependent signaling as a G protein-coupled receptor (GPCR) and also limits CCL2 signaling as a scavenger receptor. We investigated the mechanisms underlying CCR2 scavenging, including the involvement of intracellular proteins typically associated with GPCR signaling and internalization. Using CRISPR knockout cell lines, we showed that CCR2 scavenged by constitutively internalizing to remove CCL2 from the extracellular space and recycling back to the cell surface for further rounds of ligand sequestration. This process occurred independently of G proteins, GPCR kinases (GRKs), ß-arrestins, and clathrin, which is distinct from other "professional" chemokine scavenger receptors that couple to GRKs, ß-arrestins, or both. These findings set the stage for understanding the molecular regulators that determine CCR2 scavenging and may have implications for drug development targeting this therapeutically important receptor.

Mucin 2 (MUC2) modulates the aggressiveness of breast cancer.

PURPOSE: Tumors that secrete large volumes of mucus are chemotherapy resistant, however, mechanisms underlying this resistance are unknown. One protein highly expressed in mucin secreting breast cancers is the secreted mucin, Mucin 2 (MUC2). While MUC2 is expressed in some breast cancers it is absent in normal breast tissue, implicating it in breast cancer. However, the effects of MUC2 on breast cancer are largely unknown. This study examined the role of MUC2 in modulating breast cancer proliferation, response to chemotherapy and metastasis. METHODS: Using patient derived xenografts we developed two novel cell lines, called BCK4 and PT12, which express high levels of MUC2. To modulate MUC2 levels, BCK4 and PT12 cells were engineered to express shRNA targeted to MUC2 (shMUC2, low MUC2) or a non-targeting control (shCONT, high MUC2) and proliferation and apoptosis were measured in vitro and in vivo. BCK4 cells with shCONT or shMUC2 were labeled with GFP-luciferase and examined in an experimental metastasis model; disease burden and site specific dissemination were monitored by intravital imaging and fluorescence guided dissection, respectively. RESULTS: Proliferation decreased in BCK4 and PT12 shMUC2 cells versus control cells both in vitro and in vivo. Chemotherapy induced minimal apoptosis in control cells expressing high MUC2 but increased apoptosis in shMUC2 cells containing low MUC2. An experimental metastasis model showed disease burden decreased when breast cancer cells contained low versus high MUC2. Treatment with Epidermal Growth Factor (EGF) increased MUC2 expression in BCK4 cells; this induction was abolished by the EGF-receptor inhibitor, Erlotinib. CONCLUSIONS: MUC2 plays an important role in mediating proliferation, apoptosis and metastasis of breast cancer cells. MUC2 may be important in guiding treatment and predicting outcomes in breast cancer patients.

Estrogen induces c-Kit and an aggressive phenotype in a model of invasive lobular breast cancer.

Among the molecular subtypes of breast cancer are luminal (A or B) estrogen receptor positive (ER+), HER2+, and triple negative (basal-like). In addition to the molecular subtypes, there are 18 histologic breast cancer subtypes classified on appearance, including invasive lobular breast carcinoma (ILC), which are 8-15% of all breast cancers and are largely ER+ tumors. We used a new model of ER+ ILC, called BCK4. To determine the estrogen regulated genes in our ILC model, we examined BCK4 xenograft tumors from mice supplemented with or without estrogen using gene expression arrays. Approximately 3000 genes were regulated by estrogen in vivo. Hierarchical cluster analyses of the BCK4 derived tumors compared with ER+ and ER- breast cancer cell lines show the estrogen treated BCK4 tumors group with ER- breast cancers most likely due to a high proliferation score, while tumors from cellulose supplemented mice were more related to ER+ breast tumor cells. To elucidate genes regulated in vitro by estrogen in BCK4 cells, we performed expression profiling using Illumina arrays of the BCK4 cell line, treated with or without estrogen in vitro. A set of ~200 overlapping genes were regulated by estrogen in the BCK4 cell line and xenograft tumors, and pathway analysis revealed that the c-Kit pathway might be a target to reduce estrogen-induced proliferation. Subsequent studies found that inhibition of c-Kit activity using imatinib mesylate (Gleevec®) blocked estrogen mediated stimulation of BCK4 tumors and BCK4 cells in vitro as effectively as the anti-estrogen fulvestrant (Faslodex®). Decreased expression of c-Kit using shRNA also decreased baseline and estrogen induced proliferation in vitro and in vivo. These studies are the first to indicate that c-Kit inhibition is an effective approach to target c-Kit+ ILC.

Experimental models of cardiac transplantation: design determines relevance.

PURPOSE OF REVIEW: Experimental models have contributed enormously to basic immunology. However, the use of reductionist experiments has produced results that are not always successfully translated into the clinic. Recently, incorporation of more realistic clinical parameters in experimental designs has produced new insights relevant to cardiac transplantation. RECENT FINDINGS: Experiments in mice have provided crucial insights into the concept that T cell responses to pathogens generate memory cells with cross-reactive specificities for histocompatibility antigens. These memory T cells are resistant to current immunosuppressive strategies. Memory T cells infiltrate grafts within hours after transplantation, and grafts subjected to clinically relevant periods of cold ischemia are more susceptible to injury by this cellular infiltrate. Early immune responses now can be investigated with improved 'humanized' mice. Mice with multiple knock-in genes for human cytokines support development of human monocytes, macrophages and natural killer cells in increased numbers and with better function. SUMMARY: Better and more clinically relevant experimental designs are providing animal models tailored to address clinic exigencies.