CD97, an adhesion-linked G-protein-coupled receptor (GPCR), is induced in multiple epithelial cancer lineages. We address here the signaling properties and the functional significance of CD97 expression in prostate cancer. Our findings show that CD97 signals through Ga12/13 to increase RHO-GTP levels. CD97 functioned to mediate invasion in prostate cancer cells, at least in part, by associating with lysophosphatidic acid receptor 1 (LPAR1), leading to enhanced LPA-dependent RHO and extracellular signal-regulated kinase activation. Consistent with its role in invasion, depletion of CD97 in PC3 cells resulted in decreased bone metastasis without affecting subcutaneous tumor growth. Furthermore, CD97 heterodimerized and functionally synergized with LPAR1, a GPCR implicated in cancer progression. We also found that CD97 and LPAR expression were significantly correlated in clinical prostate cancer specimens. Taken together, these findings support the investigation of CD97 as a potential therapeutic cancer target. Cancer Res; 71(23); 7301-11. Ó2011 AACR.

Cse4 is posttranslationally modified in Saccharomyces cerevisiae. Ipl1 contributes to Cse4 phosphorylation in vivo and in vitro. Phosphorylation of Cse4 at centromeres is enhanced in response to nocodazole or reduced cohesion. The results suggest that phosphorylation of Cse4 ensures faithful chromosome segregation.

One great challenge in our understanding of TGF-β cancer biology and the successful application of TGF-β targeted therapy is that TGF-β works as both a tumor suppressor and a tumor promoter. The underlying mechanisms for its functional change remain to be elucidated. Using 4T1 mammary tumor model that shares many characteristics with human breast cancer, particularly its ability to spontaneously metastasize to the lungs, we demonstrate that Gr-1+CD11b+ cells or myeloid derived suppressor cells (MDSCs) are important mediators in TGF-β regulation of mammary tumor progression. Depletion of Gr-1+CD11b+ cells diminished the anti-tumor effect of TGF-β neutralization. Two mechanisms were involved: first, treatment with TGF-β neutralization antibody (1D11) significantly decreased the number of Gr-1+CD11b+ cells in tumor tissues and premetastatic lung. This is mediated through increased Gr-1+CD11b+ cell apoptosis. In addition, 1D11 treatment significantly decreased the expression of Th2 cytokines & Arginase 1. Interestingly, the number and property of Gr-1+CD11b+ cells in peripheral blood/draining lymph nodes correlated with tumor size and metastases in response to 1D11 treatment. Our data suggest that the efficacy of TGF-β neutralization depends on the presence of Gr-1+CD11b+ cells, and these cells could be good biomarkers for TGF-β targeted therapy.

Starvation of Dictyostelium induces a developmental program in which cells form an aggregate that eventually differentiates into a multicellular structure. The aggregate formation is mediated by directional migration of individual cells that quickly transition to group migration in which cells align in a head-to-tail manner to form streams. Cyclic AMP acts as a chemoattractant and its production, secretion, and degradation are highly regulated. A key protein is the extracellular phosphodiesterase PdsA. In this study we examine the role and localization of PdsA during chemotaxis and streaming. We find that pdsA ؊ cells respond chemotactically to a narrower range of chemoattractant concentrations compared with wild-type (WT) cells. Moreover, unlike WT cells, pdsA ؊ cells do not form streams at low cell densities and form unusual thick and transient streams at high cell densities. We find that the intracellular pool of PdsA is localized to the endoplasmic reticulum, which may provide a compartment for storage and secretion of PdsA. Because we find that cAMP synthesis is normal in cells lacking PdsA, we conclude that signal degradation regulates the external cAMP gradient field generation and that the group migration behavior of these cells is compromised even though their signaling machinery is intact. INTRODUCTIONThe process by which cells sense an attractant molecule and respond by migrating directionally toward it is called chemotaxis. Chemotaxis is essential for a variety of physiological processes in mammals as well as for the survival of lower eukaryotes. The mechanisms that regulate the directed migration of neutrophils and Dictyostelium discoideum have been extensively studied previously (van Haastert and Devreotes, 2004;Bagorda et al., 2006;Friedl and Weigelin, 2008;Stephens et al., 2008). One particular challenge of these fast-moving cells is to maintain the ability to respond sensitively and rapidly to an attractant signal. Because both neutrophils and Dictyostelium cells use signal relay loops to propagate chemoattractant signals, mechanisms must be at play to maintain detectable levels of chemoattractants. Remarkably, signal degradation also seems to play an important role in the developmental processes of multicellular organisms. Migration of primordial germ cells in Drosophila and Zebrafish seems to require degradation of specific lysophospholipids in the former or chemokines in the latter to allow these cells to find their proper developmental environment (Renault and Lehmann, 2006;Minina et al., 2007). Many cell types use receptor-mediated degradation to maintain detectable levels of chemoattractants. In this context, cells internalize the bound receptor and as part of the receptor recycling pathway the ligand is released for degradation (Minina et al., 2007;Boldajipour et al., 2008;Borroni et al., 2008;Scola et al., 2008). Alternatively, extracellular enzymes can specifically degrade the extracellular signaling molecules, allowing the sensing machinery to go back to basal levels and retain high se...

BackgroundProlactin is a polypeptide hormone responsible for proliferation and differentiation of the mammary gland. More recently, prolactin's role in mammary carcinogenesis has been studied with greater interest. Studies from our laboratory and from others have demonstrated that three specific isoforms of the prolactin receptor (PRLR) are expressed in both normal and cancerous breast cells and tissues. Until now, reliable isoform specific antibodies have been lacking. We have prepared and characterized polyclonal antibodies against each of the human PRLR isoforms that can effectively be used to characterize human breast cancers.MethodsRabbits were immunized with synthetic peptides of isoform unique regions and immune sera affinity purified prior to validation by Western blot and immunohistochemical analyses. Sections of ductal and lobular carcinomas were stained with each affinity purified isoform specific antibody to determine expression patterns in breast cancer subclasses.ResultsWe show that the rabbit antibodies have high titer and could specifically recognize each isoform of PRLR. Differences in PRLR isoform expression levels were observed and quantified using histosections from xenografts of established human breast cancer cells lines, and ductal and lobular carcinoma human biopsy specimens. In addition, these results were verified by real-time PCR with isoform specific primers. While nearly all tumors contained LF and SF1b, the majority (76%) of ductal carcinoma biopsies expressed SF1a while the majority of lobular carcinomas lacked SF1a staining (72%) and 27% had only low levels of expression.ConclusionsDifferences in the receptor isoform expression profiles may be critical to understanding the role of PRL in mammary tumorigenesis. Since these antibodies are specifically directed against each PRLR isoform, they are valuable tools for the evaluation of breast cancer PRLR content and have potential clinical importance in treatment of this disease by providing new reagents to study the protein expression of the human PRLR.

The Rab family of Ras-related GTPases are part of a complex signaling circuitry in eukaryotic cells, yet we understand little about the mechanisms that underlie Rab protein participation in such signal transduction networks, or how these networks are integrated at the physiological level. Reversible protein phosphorylation is widely used by cells as a signaling mechanism. Several phospho-Rabs have been identified, however the functional consequences of the modification appear to be diverse and need to be evaluated on an individual basis. In this study we demonstrate a role for phosphorylation as a negative regulatory event for the action of the yeast Rab GTPase Sec4p in regulating polarized growth. Our data suggest that the phosphorylation of the Rab Sec4p prevents interactions with its effector, the exocyst component Sec15p, and that the inhibition may be relieved by a PP2A phosphatase complex containing the regulatory subunit Cdc55p.

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Transforming growth factor-β (TGF-β) is an important regulator of cellular homeostasis and disease pathogenesis. Canonical TGF-β signaling occurs through Smad2/3–Smad4 complexes; however, recent in vitro studies suggest that elevated levels of TGF-β may activate a novel mixed Smad complex (Smad2/3-Smad1/5/9), which is required for some of the pro-oncogenic activities of TGF-β. To determine if mixed Smad complexes are evident in vivo, we developed antibodies that can be used with a proximity ligation assay to detect either canonical or mixed Smad complexes in formalin-fixed paraffin-embedded sections. We demonstrate high expression of mixed Smad complexes in the tissues from mice genetically engineered to express high levels of TGF-β1. Mixed Smad complexes were also prominent in 15–16 day gestation mouse embryos and in breast cancer xenografts, suggesting important roles in embryonic development and tumorigenesis. In contrast, mixed Smad complexes were expressed at extremely low levels in normal adult mouse tissue, where canonical complexes were correspondingly higher. We show that this methodology can be used in archival patient samples and tissue microarrays, and we have developed an algorithm to quantitate the brightfield read-out. These methods will allow quantitative analysis of cell type-specific Smad signaling pathways in physiological and pathological processes.