Suzanne McFarlane

CD44 Potentiates the Adherence of Metastatic Prostate and Breast Cancer Cells to Bone Marrow Endothelial Cells

The aim of this current study was to examine the significance of CD44 expression in mediating cancer cell adhesion to human bone marrow endothelial cell(s) (hBMEC). Differential CD44 expression on two metastatic prostate cancer cell lines, PC3 (CD44 +ve) and DU145 (CD44 −ve) and four breast cancer cell lines was confirmed by immunoblotting and immunocytochemistry. In cell adhesion assays, PC3 but not DU145 cells demonstrated a rapid adhesion to hBMECs. Treatment of PC3 cells with a neutralizing antibody against CD44 standard (CD44s) and CD44 splice variants decreased PC3 cell adhesion to hBMECs. Similarly, depletion of CD44 expression using RNA interference decreased the ability of PC3 cells and two CD44 +ve breast cancer cell lines (MDA-MB-231 and MDA-MB-157) to bind FITC-conjugated hyaluronan (FITC-HA) and to adhere to hBMECs. In contrast, transfection of DU145 cells or the T47D and MCF-7 breast cancer cell lines to express CD44s increased cell surface binding of FITC-HA and cell adherence to hBMECs. Treatment of PC3 and MDA-MD-231 cells but not hBMECs with hyaluronidase attenuated cell adhesion, suggesting that cell surface expression of CD44 on prostate and breast cancer cells may promote the retention of a HA coat that facilitates their initial arrest on bone marrow endothelium.

Cortactin underpins CD44-promoted invasion and adhesion of breast cancer cells to bone marrow endothelial cells

Using a validated tetracycline (tet)-regulated MCF7-founder (MCF7F) expression system to modulate expression of CD44 standard form (CD44s), we report the functional importance of CD44s and that of a novel transcriptional target of hyaluronan (HA)/CD44s signaling, EMS1/cortactin, in underpinning breast cancer metastasis. In functional experiments, tet-regulated induction of CD44s potentiated the migration and invasion of MCF7F cells through HA-supplemented Matrigel. EMS1/cortactin was identified by expression profiling as a novel transcriptional target of HA/CD44 signaling, an association validated by quantitative PCR and immunoblotting experiments in a range of breast cancer cell lines. The mechanistic basis underpinning CD44-promoted transcription of EMS1/cortactin was shown to be dependent upon a NFκB mechanism, since pharmacological inhibition of IκKinase-2 or suppression of p65 Rel A expression attenuated CD44-induced increases in cortactin mRNA transcript levels. Overexpression of a c-myc tagged murine cortactin construct in the weakly invasive, CD44-deficient MCF7F and T47D cells potentiated their invasion. Furthermore, the functional importance of cortactin to CD44s-promoted metastasis was demonstrated by selective suppression of cortactin in CD44-expressing MCF7F-B5 and MDA-MB-231 breast cancer cells using RNAi, which was shown to result in attenuated CD44-promoted invasion and CD44-promoted adhesion to bone marrow endothelial cells (BMECs).

Chloroquine causes lysosomal dysfunction in neural retina and RPE: Implications for retinopathy

Chronic use of chloroquine has been shown to induce numerous pathophysiological defects in the retina. This drug has the ability to alter pH of intracellular compartments and lysosomal function of the retinal pigment epithelium (RPE) and retinal neurons may constitute the basis of chloroquine retinopathy. The aim of the current study was to investigate pathogenic alterations in retinal cells continuously exposed to chloroquine using appropriate in vivo and in vitro models. Male hooded Lister rats were implanted with osmotic mini pumps which released chloroquine continuously over a period of seven days. The eyes were processed for electron microscopy and ultrastructural abnormalities determined in the neural retina and quantified using stereology in the retinal pigment epithelium (RPE). RPE were also exposed to chloroquine in vitro and lysosomal pH changes were investigated using a pH sensitive probe. Degradative capacity was also analysed using FITC labeled rod outer segments (ROS). Chloroquine-treated animals displayed several ultra-structural abnormalities including numerous membranous cytoplasmic bodies (MCBs) in retinal neurons. Cone photoreceptors displayed numerous MCBs although rods did not. The RPE of the treated groups all showed significantly higher numbers of lysosomal associated organelles (LAO) than the control group (p < 0.001). The in vitro experiments demonstrated chloroquine-mediated rises in lysosomal pH and an increase in lysosome/phagosome accumulation of ROS in the chloroquine treated group (p < 0.01). The current study demonstrates that chloroquine disrupts lysosomal function in retinal neurons and RPE. The evidence presented provides a clear pathogenic basis for the functional defects experienced by patients with chloroquine retinopathy.

Agelastatin A: a novel inhibitor of osteopontin-mediated adhesion, invasion, and colony formation

Effective inhibitors of osteopontin (OPN)–mediated neoplastic transformation and metastasis are still lacking. (-)-Agelastatin A is a naturally occurring oroidin alkaloid with powerful antitumor effects that, in many cases, are superior to cisplatin in vitro. In this regard, past comparative assaying of the two agents against a range of human tumor cell lines has revealed that typically (-)-agelastatin A is 1.5 to 16 times more potent than cisplatin at inhibiting cell growth, its effects being most pronounced against human bladder, skin, colon, and breast carcinomas. In this study, we have investigated the effects of (-)-agelastatin A on OPN-mediated malignant transformation using mammary epithelial cell lines. Treatment with (-)-agelastatin A inhibited OPN protein expression and enhanced expression of the cellular OPN inhibitor, Tcf-4. (-)-Agelastatin A treatment also reduced β-catenin protein expression and reduced anchorage-independent growth, adhesion, and invasion in R37 OPN pBK-CMV and C9 cell lines. Similar effects were observed in MDA-MB-231 and MDA-MB-435s human breast cancer cell lines exposed to (-)-agelastatin A. Suppression of Tcf-4 by RNA interference (short interfering RNA) induced malignant/invasive transformation in parental benign Rama 37 cells; significantly, these events were reversed by treatment with (-)-agelastatin A. Our study reveals, for the very first time, that (-)-agelastatin A down-regulates β-catenin expression while simultaneously up-regulating Tcf-4 and that these combined effects cause repression of OPN and inhibition of OPN-mediated malignant cell invasion, adhesion, and colony formation in vitro. We have also shown that (-)-agelastatin A inhibits cancer cell proliferation by causing cells to accumulate in the G2 phase of cell cycle. [Mol Cancer Ther 2008;7(3):548–58]

CD44 enhances invasion of basal-like breast cancer cells by upregulating serine protease and collagen- degrading enzymatic expression and activity.

IntroductionBasal-like breast cancers (BL-BCa) have the worst prognosis of all subgroups of this disease. Hyaluronan (HA) and the HA receptor CD44 have a long-standing association with cell invasion and metastasis of breast cancer. The purpose of this study was to establish the relation of CD44 to BL-BCa and to characterize how HA/CD44 signaling promotes a protease-dependent invasion of breast cancer (BrCa) cells.MethodsCD44 expression was determined with immunohistochemistry (IHC) analysis of a breast cancer tissue microarray (TMA). In vitro experiments were performed on a panel of invasive BL-BCa cell lines, by using quantitative polymerase chain reaction (PCR), immunoblotting, protease activity assays, and invasion assays to characterize the basis of HA-induced, CD44-mediated invasion.ResultsExpression of the hyaluronan (HA) receptor CD44 associated with the basal-like subgroup in a cohort of 141 breast tumor specimens (P = 0.018). Highly invasive cells of the representative BL-BCa cell line, MDA-MB-231 (MDA-MB-231Hi) exhibited increased invasion through a basement membrane matrix (Matrigel) and collagen. In further experiments, HA-induced promotion of CD44 signaling potentiated expression of urokinase plasminogen activator (uPA) and its receptor uPAR, and underpinned an increased cell-associated activity of this serine protease in MDA-MB-231Hi and a further BL-BCa cell line, Hs578T cells. Knockdown of CD44 attenuated both basal and HA-stimulated uPA and uPAR gene expression and uPA activity. Inhibition of uPA activity by using (a) a gene-targeted RNAi or (b) a small-molecule inhibitor of uPA attenuated HA-induced invasion of MDA-MB-231Hi cells through Matrigel. HA/CD44 signaling also was shown to increase invasion of MDA-MB-231 cells through collagen and to potentiate the collagen-degrading activity of MDA-MB-231Hi cells. CD44 signaling was subsequently shown to upregulate expression of two potent collagen-degrading enzymes, the cysteine protease cathepsin K and the matrix metalloprotease MT1-MMP. RNAi- or shRNA-mediated depletion of CD44 in MDA-MB-231Hi cells decreased basal and HA-induced cathepsin K and MT1-MMP expression, reduced the collagen-degrading activity of the cell, and attenuated cell invasion through collagen. Pharmacologic inhibition of cathepsin K or RNAi-mediated depletion of MT1-MMP also attenuated MDA-MB-231Hi cell invasion through collagen.ConclusionHA-induced CD44 signaling increases a diverse spectrum of protease activity to facilitate the invasion associated with BL-BCa cells, providing new insights into the molecular basis of CD44-promoted invasion.

Characterisation of the advanced glycation endproduct receptor complex in the retinal pigment epithelium

Aims: Advanced glycation endproducts (AGEs) accumulate with ageing and may have a significant impact on age related dysfunction of the retinal pigment epithelium (RPE). Many of the cellular effects of AGEs in other cell types are mediated through AGE binding proteins. The aim of this study was to characterise the AGE receptor complex in RPE cells in vitro and to focus on the role of the R3 component (galectin-3) as the primary effector of the complex. Methods: Primary cultures of bovine RPE cells and the human D407 RPE cell line were exposed to AGE modified albumin. Receptor expression was determined using mRNA analysis by quantitative real time RT-PCR and protein characterisation by western blotting. Immunocytochemical analysis examined the cellular localisation of the various components of the AGE receptor complex. The role of the galectin-3 receptor component was examined by transfection and overexpression using the D407 cell line and analysis of soluble AGE-R3 by ELISA. Results: All three components of the AGE receptor complex were expressed by bovine and human RPE cells. AGE exposure upregulated two components of the receptor complex and also induced significant RPE expression of VEGF mRNA (p<0.05). RPE D407 cells stably transfected to overexpress galectin-3 showed less VEGF induction. In non-transfected RPE which were exposed to AGEs, there was less soluble galectin-3 protein released into the medium (p<0.05), a response that was not evident in transfected cells. Conclusion: A conserved AGE receptor complex is evident in primary cultures of bovine RPE cells and also in a human cell line. These cells show a pathological response to AGE exposure, an effect which appears to be modulated by the galectin-3 component of the receptor complex.

Ran GTPase in Nuclear Envelope Formation and Cancer Metastasis

Ran is a small ras-related GTPase that controls the nucleocytoplasmic exchange of macromolecules across the nuclear envelope. It binds to chromatin early during nuclear formation and has important roles during the eukaryotic cell cycle, where it regulates mitotic spindle assembly, nuclear envelope formation and cell cycle checkpoint control. Like other GTPases, Ran relies on the cycling between GTP-bound and GDP-bound conformations to interact with effector proteins and regulate these processes. In nucleocytoplasmic transport, Ran shuttles across the nuclear envelope through nuclear pores. It is concentrated in the nucleus by an active import mechanism where it generates a high concentration of RanGTP by nucleotide exchange. It controls the assembly and disassembly of a range of complexes that are formed between Ran-binding proteins and cellular cargo to maintain rapid nuclear transport. Ran also has been identified as an essential protein in nuclear envelope formation in eukaryotes. This mechanism is dependent on importin-β, which regulates the assembly of further complexes important in this process, such as Nup107-Nup160. A strong body of evidence is emerging implicating Ran as a key protein in the metastatic progression of cancer. Ran is overexpressed in a range of tumors, such as breast and renal, and these perturbed levels are associated with local invasion, metastasis and reduced patient survival. Furthermore, tumors with oncogenic KRAS or PIK3CA mutations are addicted to Ran expression, which yields exciting future therapeutic opportunities.

CD44-mediated activation of α5β1-integrin, cortactin and paxillin signaling underpins adhesion of basal-like breast cancer cells to endothelium and fibronectin-enriched matrices.

CD44 expression is elevated in basal-like breast cancer (BLBC) tissue, and correlates with increased efficiency of distant metastasis in patients and experimental models. We sought to characterize mechanisms underpinning CD44-promoted adhesion of BLBC cells to vascular endothelial monolayers and extracellular matrix (ECM) substrates. Stimulation with hyaluronan (HA), the native ligand for CD44, increased expression and activation of β1-integrin receptors, and increased α5-integrin subunit expression. Adhesion assays confirmed that CD44-signalling potentiated BLBC cell adhesion to endothelium and Fibronectin in an α5B1-integrin-dependent mechanism. Co-immunoprecipitation experiments confirmed HA-promoted association of CD44 with talin and the β1-integrin chain in BLBC cells. Knockdown of talin inhibited CD44 complexing with β1-integrin and repressed HA-induced, CD44-mediated activation of β1-integrin receptors. Immunoblotting confirmed that HA induced rapid phosphorylation of cortactin and paxillin, through a CD44-dependent and β1-integrin-dependent mechanism. Knockdown of CD44, cortactin or paxillin independently attenuated the adhesion of BL-BCa cells to endothelial monolayers and Fibronectin. Accordingly, we conclude that CD44 induced, integrin-mediated signaling not only underpins efficient adhesion of BLBC cells to BMECs to facilitate extravasation but initiates their adhesion to Fibronectin, enabling penetrant cancer cells to adhere more efficiently to underlying Fibronectin-enriched matrix present within the metastatic niche.

CD44 signalling increases cathepsin K and MT1MMP expression to potentiate breast cancer cell invasion through collagen I

C28 The hyaluronan (HA) receptor CD44 is implicated in the tumourigenicity and metastasis of breast cancer. Using a tetracycline-regulated CD44 expression system in the MCF-7F breast cancer cell line, we have shown that CD44-HA signalling increases mRNA transcripts levels and protein expression of the cysteine protease cathepsin K (CatK) and the matrix metalloprotease MT1-MMP using quantitative PCR (qPCR), immunoblotting and/or ELISA. These enzymes are implicated in the proteolysis of collagen I, a major component of the bone matrix whose degradation is a major consequence of osteolytic metastasis of breast cancer. Experiments conducted using parental and a bone-homing clone of the MDA-MB-231 cell line (MDA-MB-231BO) have shown that CD44, CatK and MT1-MMP expression is elevated in the MDA-MB-231BO cells relative to the parental cells. Furthermore, CD44-HA signalling was shown to increase CatK and MT1-MMP expression in the MDA-MB-231BO cells. Consistent with their increased metastatic phenotype, MDA-MB-231BO cells displayed an enhanced invasion on HA-supplemented Matrigel™ and collagen I and demonstrated elevated collagenolytic activity as demonstrated using an in vitro fluorescence based assay. RNAi-mediated depletion of CD44 and MT1-MMP expression and pharmacological inhibition of CatK attenuated CD44-promoted invasion through a collagen I matrix. Our studies demonstrate that CD44 signalling regulates collagenase activity in breast cancer cells underpinning their invasion through matrix substrates that are enriched within breast tissue and organs to which this disease preferentially metastasizes. The long term objective of our research will be to determine whether CD44 expression and that of its transcriptional targets may be predictive for those breast cancer patients at higher risk of developing skeletal disease and/or potentially lead to the development of novel and more effective therapeutic strategies to attenuate bone metastasis.

Characterization of a cytoskeletal signaling pathway underpinning CD44-initiated, integrin-mediated adhesion of breast cancer cells to bone marrow endothelium

C29 CD44 potentiates the primary adhesion of breast cancer cells to human bone marrow endothelial cells (BMECs). The aim of this study was to establish the distal events underpinning CD44-initiated adhesion, using parental and bone-homing (BO) clones of the MDA-MB-231 breast cancer cell line. MDA-MB-231BO cells displayed increased CD44 expression and adhesion to both BMECs and fibronectin, relative to parental cells. MDA-MB-231BO cells also displayed increased expression and activation of the β1-integrin subunit. In addition the bone homing cells exhibited elevated constitutive phosphorylation of the kinases Src and FAK and the cytoskeletal proteins cortactin and paxillin relative to the parental cells. Stimulation of MDA-MB-231BO cells with the CD44 ligand hyaluronan (HA) induced an increase in the expression of the β1-integrin chain, FAK and paxillin and furthermore, promoted a rapid increase in the activation status of the β1-integrin subunits, Src, cortactin and paxillin in these cells. The HA-induced phosphorylation of paxillin was attenuated by depletion of CD44 and cortactin expression using selective RNAi strategies, suggesting that it is a downstream target of HA-CD44-cortactin signaling. MDA-MB-231BO cell adhesion to fibronectin or to hBMECs was attenuated by RNAi-mediated suppression of CD44, cortactin and paxillin expression or following administration of two neutralizing antibodies that inhibit β1-integrin and α4β1-integrin receptor signaling. Antibody-based inhibition of integrin signaling also attenuated the HA-induced phosphorylation of cortactin and paxillin suggesting that these proteins constitute a signaling cascade activated downstream of a CD44-initiated, integrin-dependent process. Our results describe a molecular pathway promoting cytoskeletal reorganization that is activated downstream of a CD44 induced, integrin-dependent event and which is critical to efficient breast cancer cell adhesion to hBMECs.