Marcia L. Graves

Toll-like receptor ligands sensitize B-cell receptor signalling by reducing actin-dependent spatial confinement of the receptor

Integrating signals from multiple receptors allows cells to interpret the physiological context in which a signal is received. Here we describe a mechanism for receptor crosstalk in which receptor-induced increases in actin dynamics lower the threshold for signalling by another receptor. We show that the Toll-like receptor ligands lipopolysaccharide and CpG DNA, which are conserved microbial molecules, enhance signalling by the B-cell antigen receptor (BCR) by activating the actin-severing protein cofilin. Single-particle tracking reveals that increased severing of actin filaments reduces the spatial confinement of the BCR within the plasma membrane and increases BCR mobility. This allows more frequent collisions between BCRs and greater signalling in response to low densities of membrane-bound antigen. These findings implicate actin dynamics as a means of tuning receptor signalling and as a mechanism by which B cells distinguish inert antigens from those that are accompanied by indicators of microbial infection.

The anti-adhesive mucin podocalyxin may help initiate the transperitoneal metastasis of high grade serous ovarian carcinoma

High grade serous ovarian tumors often metastasize transperitoneally, a process that begins when small tumor nodules de-adhere and are released into the fluid of the abdominal cavity where they float freely to reach new sites on the peritoneal wall. Podocalyxin, a small anti-adhesive sialomucin, has been shown to contribute to non-adhesive membrane domain formation in some epithelia and is overexpressed in a variety of cancers. We therefore assessed podocalyxin expression on a previously characterized tissue microarray and found that 87% (169/194) of high grade serous epithelial ovarian carcinomas were positive for podocalyxin. In addition, cell surface localization of podocalyxin was associated with a significant decrease in disease-free survival in these tumors. When podocalyxin was force-expressed in serous ovarian carcinoma-derived OVCAR-3 cells it was targeted to the cell surface and it decreased the adhesion of these cells to mesothelial monolayers, fibronectin and immobilized β1 integrin-binding antibodies. This decrease in adhesion was associated with a modest decrease in cell surface β1 integrin. In monolayer culture, podocalyxin was targeted to the free, apical domains of OVCAR-3 cells and it appeared to decrease β1 integrin levels on the attached basolateral domains of the same cells. Furthermore, in 3-dimensional basement membrane gel culture, the cells formed small, cohesive nodules and podocalyxin localized to membrane domains at the cell–basement membrane interface. Therefore, podocalyxin’s ability to facilitate the formation of non-adhesive membrane domains may contribute to the formation of free-floating high grade serous tumor nodules during the initial steps of transperitoneal metastasis.

Podocalyxin enhances breast tumor growth and metastasis and is a target for monoclonal antibody therapy

IntroductionPodocalyxin (gene name PODXL) is a CD34-related sialomucin implicated in the regulation of cell adhesion, migration and polarity. Upregulated expression of podocalyxin is linked to poor patient survival in epithelial cancers. However, it is not known if podocalyxin has a functional role in tumor progression.MethodsWe silenced podocalyxin expression in the aggressive basal-like human (MDA-MB-231) and mouse (4T1) breast cancer cell lines and also overexpressed podocalyxin in the more benign human breast cancer cell line, MCF7. We evaluated how podocalyxin affects tumorsphere formation in vitro and compared the ability of podocalyxin-deficient and podocalyxin-replete cell lines to form tumors and metastasize using xenogenic or syngeneic transplant models in mice. Finally, in an effort to develop therapeutic treatments for systemic cancers, we generated a series of antihuman podocalyxin antibodies and screened these for their ability to inhibit tumor progression in xenografted mice.ResultsAlthough deletion of podocalyxin does not alter gross cell morphology and growth under standard (adherent) culture conditions, expression of PODXL is required for efficient formation of tumorspheres in vitro. Correspondingly, silencing podocalyxin resulted in attenuated primary tumor growth and invasiveness in mice and severely impaired the formation of distant metastases. Likewise, in competitive tumor engraftment assays where we injected a 50:50 mixture of control and shPODXL (short-hairpin RNA targeting PODXL)-expressing cells, we found that podocalyxin-deficient cells exhibited a striking decrease in the ability to form clonal tumors in the lung, liver and bone marrow. Finally, to validate podocalyxin as a viable target for immunotherapy, we screened a series of novel antihuman podocalyxin antibodies for their ability to inhibit tumor progression in vivo. One of these antibodies, PODOC1, potently blocked tumor growth and metastasis.ConclusionsWe show that podocalyxin plays a key role in the formation of primary tumors and distant tumor metastasis. In addition, we validate podocalyxin as potential target for monoclonal antibody therapy to inhibit primary tumor growth and systemic dissemination.

Selective pharmacological inhibition of phosphoinositide 3-kinase p110delta opposes the progression of autoimmune diabetes in non-obese diabetic (NOD) mice

During the progression of autoimmune (type 1) diabetes, T cells and macrophages infiltrate the pancreas, disrupt islet function, and destroy insulin-producing beta cells. B-lymphocytes, particularly innate like B-cell populations such as marginal zone B cells and B-1 cells, have been implicated in many autoimmune diseases, and non-obese diabetic (NOD) mice that lack B cells do not develop spontaneous autoimmune diabetes. Hence, inhibitors of B cell signaling pathways could be useful for limiting the autoimmune processes that contribute to type 1 diabetes. Signaling via phosphoinositide 3-kinase (PI3K) regulates many cellular processes. The p110δ isoform of PI3K is expressed primarily in cells of hematopoietic origin and the catalytic activity of p110δ is important for B cell migration, activation, proliferation, and antigen presentation. Because innate-like B cells are particularly sensitive to inhibition of p110δ activity, and p110δ inhibitors also suppress pro-inflammatory functions of other cell types that contribute to autoimmunity, we tested whether a p110δ inhibitor could delay the onset or reduce the incidence of autoimmune diabetes in NOD mice. We found that long-term preventative treatment of pre-diabetic NOD mice with IC87114, a highly selective small molecule inhibitor of p110δ, reduced the infiltration of inflammatory cells into the pancreatic islets and, accordingly, delayed and reduced the loss of glucose homeostasis. Moreover in a therapeutic treatment mode, IC87114 treatment conferred prolonged protection from progression to overt diabetes in a number of animals. These findings suggest that PI3Kδ inhibitors could be useful for managing type 1 diabetes.

The cell surface mucin podocalyxin regulates collective breast tumor budding.

BackgroundOverexpression of the transmembrane sialomucin podocalyxin, which is known to play a role in lumen formation during polarized epithelial morphogenesis, is an independent indicator of poor prognosis in a number of epithelial cancers, including those that arise in the breast. Therefore, we set out to determine if podocalyxin plays a functional role in breast tumor progression.MethodsMCF-7 breast cancer cells, which express little endogenous podocalyxin, were stably transfected with wild type podocalyxin for forced overexpression. 4T1 mammary tumor cells, which express considerable endogenous podocalyxin, were retrovirally transduced with a short hairpin ribonucleic acid (shRNA) targeting podocalyxin for stable knockdown. In vitro, the effects of podocalyxin on collective cellular migration and invasion were assessed in two-dimensional monolayer and three-dimensional basement membrane/collagen gel culture, respectively. In vivo, local invasion was assessed after orthotopic transplantation in immunocompromised mice.ResultsForced overexpression of podocalyxin caused cohesive clusters of epithelial MCF-7 breast tumor cells to bud off from the primary tumor and collectively invade the stroma of the mouse mammary gland in vivo. This budding was not associated with any obvious changes in histoarchitecture, matrix deposition or proliferation in the primary tumour. In vitro, podocalyxin overexpression induced a collective migration of MCF-7 tumor cells in two-dimensional (2-D) monolayer culture that was dependent on the activity of the actin scaffolding protein ezrin, a cytoplasmic binding partner of podocalyxin. In three-dimensional (3-D) culture, podocalyxin overexpression induced a collective budding and invasion that was dependent on actomyosin contractility. Interestingly, the collectively invasive cell aggregates often contained expanded microlumens that were also observed in vivo. Conversely, when endogenous podocalyxin was removed from highly metastatic, but cohesive, 4T1 mammary tumor cells there was a decrease in collective invasion in three-dimensional culture.ConclusionsPodocalyxin is a tumor cell-intrinsic regulator of experimental collective tumor cell invasion and tumor budding.

Regulation of the BRCA1 promoter in ovarian surface epithelial cells and ovarian carcinoma cells

As BRCA1 expression is often suppressed in sporadic ovarian carcinoma we characterized the regulation of the 231nt proximal ‘L6’ fragment of the BRCA1 promoter in two human ovarian surface epithelial cell and two sporadic ovarian carcinoma cell lines. Two individual regulatory elements within L6, the ‘RIBS’ element and the potential ‘CRE’ element were each necessary, but alone not sufficient for L6 activation in all four cell lines. The latter element showed some affinity for the CREB transcription factor, but cAMP pathway stimulation failed to promote its activation. This element did, however, interact with, and was activated by, c‐Jun and Fra2 which suggests that it can interact with AP1‐like transcription factors and that it may act co‐operatively with RIBS‐binding factors to regulate BRCA1 transcription in ovarian cells.

Microenvironmental Regulation of BRCA1 Gene Expression by c-Jun and Fra2 in Premalignant Human Ovarian Surface Epithelial Cells

Reduced BRCA1 gene expression is common in the sporadic form of ovarian carcinoma. The spread of this highly lethal cancer often begins when tumor cell clusters are shed into the fluid of the abdominopelvic cavity such that they can float freely before seeding distant sites on the peritoneal walls and organs. Thus, the microenvironment that tumor cells find themselves in changes dramatically during these early shedding and floating stages of transperitoneal metastasis. To mimic this microenvironmental change in vitro, we released premalignant human ovarian surface epithelial cells from the substratum and forced them to cluster in suspension. Under these conditions, steady state levels of BRCA1 mRNA and protein fell significantly and the transcriptional activation state of the BRCA1 promoter was suppressed. Analysis of the promoter indicated that the previously identified “CRE” element located within the “positive regulatory region” (PRR) contributed to this suppression. More specifically, we show that the suppression was mediated, at least in part, by a suspension culture–driven decrease in the levels of two members of the AP1 transcription factor complex, c-Jun and Fra2, that bind to the CRE element. Therefore, a microenvironmental change that is manifested during the initial stages of ovarian carcinoma dissemination may, potentially, help suppress BRCA1 expression in sporadic tumors and thus promote their progression. Mol Cancer Res; 11(3); 272–81. ©2013 AACR.

Podocalyxin in the diagnosis and treatment of cancer

Kelly M. McNagny, Michael R. Hughes, Marcia L. Graves, Erin J. DeBruin, Kimberly Snyder, Jane Cipollone, Michelle Turvey, Poh C. Tan, Shaun McColl and Calvin D. Roskelley

Applied stretch initiates directional invasion through the action of Rap1 GTPase as a tension sensor

ABSTRACT Although it is known that a stiffening of the stroma and the rearrangement of collagen fibers within the extracellular matrix facilitate the movement of tumor cells away from the primary lesion, the underlying mechanisms responsible are not fully understood. We now show that this invasion, which can be initiated by applying tensional loads to a three-dimensional collagen gel matrix in culture, is dependent on the Rap1 GTPases (Rap1a and Rap1b, referred to collectively as Rap1). Under these conditions Rap1 activity stimulates the formation of focal adhesion structures that align with the tensional axis as single tumor cells move into the matrix. These effects are mediated by the ability of Rap1 to induce the polarized polymerization and retrograde flow of actin, which stabilizes integrins and recruits vinculin to preformed adhesions, particularly those near the leading edge of invasive cells. Rap1 activity also contributes to the tension-induced collective invasive elongation of tumor cell clusters and it enhances tumor cell growth in vivo. Thus, Rap1 mediates the effects of increased extracellular tension in multiple ways that are capable of contributing to tumor progression when dysregulated. Summary: Tension applied through 3D matrices directs invasion of tumor cells along the tensional axis. Rap1 senses the tension and polarizes the cytoskeletal and subsequent adhesion remodeling.

Abstract P1-05-05: Podocalyxin is a key regulator of breast cancer progression and metastasis.

Podocalyxin (gene name Podxl) is a CD34-related sialomucin that has been shown to be important in regulating cell adhesion, migration, and polarity of hematopoietic progenitors and vascular endothelia. In breast cancer, we have shown that podocalyxin is overexpressed on a subset of node-negative tumors and its expression is strongly associated with poor overall survival. To determine whether podocalyxin directly regulates breast cancer cell behavior, we ectopically expressed podocalyxin in the human breast cancer cell line, MCF7, which expresses low levels of endogenous podocalyxin, is minimally invasive, and non-metastatic. Upon ectopic expression of podocalyxin, MCF7 “bulge apically”, produce apical and lateral microvilli, are less adhesive in vitro, and are delayed in targeting integrins to the basolateral surface. In contrast to MCF7, MDA-MB-231 is a basal-like breast cancer cell-line, which expresses high levels of endogenous podocalyxin, exhibits poorly polarized monolayer and mammosphere architecture in vitro and forms “metastatic” lung tumors in vivo. Using Podxl-targeted shRNA vectors we show that expression of podocalyxin is required for MDA-MB-231 cells to form distant metastases in a competitive in vivo assay utilizing humanized NOD/SCIDIL-2rγ−/− (NSG) mice. Previously, we observed that Podxl-deficient hematopoietic cells have impaired CXCR4-mediated chemotaxis to CXCL12, a known axis for tumor metastasis. Our current hypothesis is that podocalyxin regulates chemotaxis of tumor-initiating cells (TICs) to the CXCL12-rich tissues of the lungs, liver, and bone marrow. Up-regulation of podocalyxin on breast cancer cells may be a key event in tumor progression and likely enables these cells to sense chemokines and metastasize to peripheral sites. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-05.