AQP1 is not only a water channel: It contributes to cell migration through Lin7/β-catenin

AQPs are water channel proteins. In particular, AQP1 was demonstrated to be involved in cell migration. According to the model proposed by Verkman and collaborators, AQP drives water influx, facilitating lamellipodia extension and cell migration. Investigating the possible connection between AQP1 and cytoskeleton, our group showed that such a water channel through Lin7/β-catenin affects the organization of the cytoskeleton and proposed a model.All together, these data appear particularly intriguing since the use of AQP1 as target might be useful to modulate angiogenesis/vasculogenic mimicry.Key words: AQP, cytoskeleton, Lin7, β-catenin, motility, molecular adhesionAquaporins were discovered by Peter Agre, who won the Nobel Prize in Chemistry in 2003. They are a family of water-specific, membrane-channel proteins expressed in diverse tissues. Two functional groups of mammalian aquaporins are now recognized: aquaporins (AQP1, AQP2, AQP4, AQP5 and AQP8) which are primarily water selective and aquaglyceroporins (AQP3, AQP7, AQP9 and AQP10) which are permeable to small uncharged solutes such as lactate, glycerol and urea in addition to water.¹ The characterization of the organization of aquaporin genes and identification of their position within the human and mouse genomes have established a primary role for some aquaporins in clinical disorders such as congenital cataracts and nephrogenic diabetes insipidus.² More recently, in the control of fat accumulation, aquaporins were demonstrated to play an important role.^3–6 A characterization of AQPs was recently carried out in neuronal stem cells.⁷ More interesting, an impairment of endothelial cell migration, without altering their proliferation or adhesion, was shown by AQP1 null mice.⁸ Based on findings of slowed lamellipodial dynamics in AQP deficiency and AQP polarization to the leading edge of migrating cells, a mechanism of AQP-facilitated cell migration was proposed by Verkman and collaborators.⁹ According to this model, actin cleavage and ion uptake at the tip of lamellipodium creates local osmotic gradients and drives water influx, facilitating lamellipodial extension and cell migration.⁹ AQP-facilitated cell migration has also been found in brain astroglial cells,^10,11 kidney proximal tube cells¹² and skin cells.¹³ In this connection, AQP1 has been proposed as a novel promoter of tumor angiogenesis.¹⁴ It is still unclear, however, how actin is cleaved. On the other hand, according to Verkman’s model, AQP1 is the water channel that drives water influx.We have recently proposed a new model. In a recent paper published in PLoS ONE Journal, we have investigated the possi-ble relationship between AQP1 and the cytoskeleton in endothelial and melanoma cells (both expressing AQP1), focusing on the possible involvement of Lin proteins.¹⁵ The latter are plasma membrane-associated proteins containing one or several PDZ domains¹⁶ and are required for the organization of the cytoskeleton. A scaffold complex common for epithelial and neuronal cells is the heterotrimeric complex consisting of the CASK/Lin-2, Lin-7 and Lin-10 PDZ proteins.^17–20 In mammals, Lin-7 can recruit cell adhesion molecules, receptors, ion channels and signaling proteins.^17–20 Therefore, heterotrimeric PDZ complex plays a role in regulating the localization of interacting proteins. The novelties of our paper are the following: firstly, AQP1 plays the same role in human melanoma and endothelial cells, suggesting that this water channel has a global physiological role. Secondly, AQP1 interacts, at least, with Lin-7/β-catenin. Another interesting aspect is that the knock down of AQP1 induced the proteolytic degradation of Lin7/β-catenin through proteasoma complex. In the model proposed in PLoS ONE Journal, AQP1 is not only a water channel but a critical scaffold for plasma-membrane associated multiprotein-complex important for cytoskeleton build-up, adhesion and motility.¹⁵ Our data show, actually, that AQP1 plays a role in stabilizing the cytoskeleton affecting the migration capacity.²¹ Considering both Verkman’s model and our findings, I suggest that, in presence of local osmotic gradients like as at the tip of lamelllipodium, water is driven inside through AQP(s), leading to the disruption of scaffold proteins which are degraded through proteasoma (Lin7/β-catenin). The effect on the cell is the cleavage of actin.These findings corroborate the analysis of manifold cellular functions of AQPs in normal cells and in diseases and the possi-bility to consider aquaporins as specific therapeutic targets for various pathophysiological conditions.²² In particular, AQP1 might be an interesting target for tumors. In fact, AQP1 is expressed both by tumor and endothelial cells and a targeted inhibition or silencing of such a protein might affect both the migratory and the angiogenesis/vasculogenic mimicry capacity.Vasculogenic mimicry was described for the first time by the unique ability of aggressive melanoma cells to express an endothelial phenotype and to form vessel-like networks in three dimensional cultures, “mimicking” the pattern of embryonic vascular networks and recapitulating the patterned networks seen in patients with aggressive tumors correlated with poor prognosis (reviewed in ref. ²³). In fact, the word “vasculogenic” was selected to indicate the generation of the pathway de novo and “mimicry” was used because the tumor uses cell pathways for transporting fluid in tissues that were clearly not blood vessels. Additional studies have reported vasculogenic mimicry in several other tumor types (reviewed in ref. ²³). As shown in Figure 1, human melanoma cell line WM115 expresses AQP1 at the plasma membrane in vitro and only a few cells express such a water channel in tumor xenograft according to the low/undetectable number of initiating/cancer stem cells found in tumor xenograft and melanoma biopsies.²⁴Open in a separate windowFigure 1(A) Immunofluorescence of AQP1 in WM115 cells. Subconfluent cells were grown on 400 mm² glass cover and fixed in 4% paraformaldeide for 20 min. Thus, the cells were incubated with the same buffer containing 0.5% TRITON X-100 for 5 min and incubated in 1% BSA-PBS for 20 min and with the primary antibody (anti-AQP-1) overnight at 4°C. A secondary antibody conjugated with TRIC was used. Nuclei were stained with DAPI. (B) 5 × 10⁴ living WM115 cells were injected subcutaneous in SCID mice and the tumor mass was collected 19 days after and processed for immunohistochemistry. The slides (4–5 mm) deparaffinized were incubated overnight with anti-AQP1 (1:100) and the colour was developed using VIP. Magnification 200X. E, endothelial cells AQP1-positive. (C) Shows a higher magnification of a part of figure shown in (B) where few melanoma cells are AQP1-positive (1,000X).Finally, there are no AQP inhibitors reported that are suitable candidates for clinical development. An interesting new way might be to study the possibility of functionally significant AQP polymorphisms. In this connection, AQP4 polymorphisms was found to be associated with increased severity of brain edema.²⁵ It may be worthwhile to investigate polymorphisms of AQP1 or other AQPs in cancer and endothelial associated tumor.     

Endothelial origin of mesenchymal stem cells

Mesenchymal stem/stromal cells (MSCs) are fibroblastoid cells capable of long-term expansion and skeletogenic differentiation. While MSCs are known to originate from neural crest and mesoderm, immediate mesodermal precursors that give rise to MSCs have not been characterized. Recently, using human embryonic stem cells (hESCs), we demonstrated that mesodermal MSCs arise from APLNR⁺ precursors with angiogenic potential, mesenchymoangioblasts, which can be identified by FGF2-dependent colony-forming assay in serum-free semisolid medium. In this overview we provide additional insights on cellular pathways leading to MSC establishment from mesoderm, with special emphasis on endothelial-mesenchymal transition as a critical step in MSC formation. In addition, we highlight an essential role of FGF2 in induction of angiogenic cells with potential to transform into MSCs (mesenchymoangioblasts) or hematopoietic cells (hemangioblasts) from mesoderm, and discuss correlations of our in vitro findings with the course of angioblast development during embryogenesis.Key words: mesenchymoangioblast, hemangioblast, human embryonic stem cells, endothelial-mesenchymal transition, epithelial-mesenchymal transition, mesenchymal stem cells, endothelial cells, apelin receptor, FGFMesenchymal stem/stromal cells (MSCs) are defined as multipotent fibroblastoid cells that give rise to cells of the skeletal connective tissue including osteoblasts, chondrocytes and adipocytes.^1–4 Although MSCs were described more than 40 years ago and are widely used for cellular therapies, very little knowledge exists regarding the developmental origins of MSCs in the embryo, the hierarchy of MSC progenitors or heterogeneity of MSCs within tissues. It has been demonstrated that during embryonic development, MSCs arise from a two major sources: neural crest and mesoderm.^5–7 Using Cre-recombinase lineage tracing experiments, Takashima et al. identified Sox1⁺ neuroepithelium as pre-cursors of MSCs of neural crest origin. However, direct precursors of mesoderm-derived MSCs were unknown. To identify these precursors, we employed human embryonic stem cells (hESCs) directed toward mesendodermal differentiation in coculture with mouse bone marrow stromal cells OP9,⁸ using the experimental approach depicted in Figure 1. As shown in this differentiation system, mesoderm reminiscent of lateral plate/extraembryonic mesoderm in the embryo can be identified by expression of apelin receptor (APLNR), otherwise known as angiotensin receptor like-1 receptor. Because we observed a positive selective effect of FGF2 on production of mesenchymal cells from hESCs in OP9 coculture, we decided to test whether FGF2 can induce the formation of colonies with mesenchymal potential from APLNR⁺ mesodermal cells. Indeed, when we isolated APLNR⁺ cells from hESCs differentiated on OP9 for 2 days and placed them in serum-free semisolid medium containing FGF2, we observed the formation of sharply-circumscribed spheroid colonies formed by tightly packed cells with a gene expression profile representative of embryonic mesenchyme originating from lateral plate/extraembryonic mesoderm and CD140a⁺CD146⁺C D90⁺CD56⁺CD166⁺CD31⁻CD43⁻CD45⁻ phenotype typical of mesenchymal cells. Based on cellular composition, we designated these colonies as mesenchymal (MS) colonies and cells forming these colonies as MS colony-forming cells (MS-CFCs). MS colony formation required serum-free medium and was solely dependent on FGF2 as a colony-forming factor. MS colonies were significantly enhanced by PDGF-BB, but suppressed by VEGF, TGFβ1 and Activin A. When transferred to the adherent cultures in serum-free medium with FGF2, individual MS colonies gave rise to multi-potential mesenchymal cell lines with typical phenotype (CD146⁺ CD105⁺ CD73⁺ CD31⁻ CD43/45⁻), differentiation (chondro-, osteo- and adipogenesis) and robust proliferation (>80 doublings) potentials. Using single cell deposition assay, chimeric hESC lines and time-lapse studies we demonstrated the clonality/single cell origin of MS colonies.Open in a separate windowFigure 1Schematic diagram of the experimental approach used to identify precursors and cellular events leading to formation of mesoderm-derived MSCs. hESCs were committed to mesendodermal differentiation through coculture with OP9 for 2 days. APLNR⁺ mesodermal cells were selected using magnetic sorting. In serum-free semisolid medium, APLNR⁺ cells grew into FGF2-dependent compact spheroid colonies composed of mesenchymal cells. MS colonies were formed through establishment of tightly-packed single cell-derived cores (day 3 of clonogenic culture), which expanded into spheroid colonies (days 6 and 12 of clonogenic culture). To evaluate differentiation potential, MS colonies were collected at different stages of clonogenic culture and placed on OP9. The presence of endothelial and mesenchymal cells after coculture of MS colonies with OP9 was evaluated by flow cytometry and immunofluorescence. In addition, colonies at core stage (day 3 of clonogenic culture) and mature colonies (day 12 of clonogenic cultures) were collected for molecular profiling studies. To generate clonal MSC lines, individual mature colonies were plated on the collagen/fibronectin-coated plastic and cultured in presence of FGF2.MS-CFCs could be detected only transiently, with a major peak on day 2 of hESC differentiation and disappeared after 4 days of differentiation. Notably, MS-CFC activity was developed prior to the expression of CD73 and CD105 MSC markers and upregulation of MSC-related genes, i.e., before onset of mesenchymogenesis. APLNR⁺ cells isolated from hESC cultures differentiated for 3 days also formed colonies in response to FGF2; however, the vast majority of these colonies were composed of blood cells and had a morphology similar to the previously described blast (BL) or hemangioblast colonies, which identify a common precursor for hematopoietic and endothelial cells.^9,10To fully evaluate the differentiation potential of MS colonies, we collected these colonies from semisolid cultures and placed them back on OP9 feeders, which are known to support development of a broad range of mesodermal lineage cells, including hematopoietic, vascular and cardiac.^11–13 Using this approach, we confirmed that individual BL colonies possess hemangioblastic potential, i.e., generate both hematopoietic and endothelial cells. When MS colonies were picked from clonogenic cultures and cultured on OP9, we found that the majority of cells differentiated into CD146⁺CD31⁻CD43/CD45⁻ mesenchymal cells as expected. However, we also discovered that MS colonies gave rise to CD31/VE-cadherin⁺CD43/45⁻ endothelial cells, indicating that MS colonies similar to BL colonies possess endothelial potential. The endothelial potential of MS colonies was also confirmed by demonstration of tube formation by MS colonies grown on Matrigel. In contrast, MSC lines derived from MS colonies did not produce any endothelial cells after coculture with OP9 indicating a progressive restriction of differentiation potential following MSC formation. Because single MS-CFC shows potential to form endothelium and MSCs, we designated the MSC precursor identified by this colony-forming assay as mesenchymoangioblast.To define more precisely the cellular events leading to establishing MSCs, we examined the formation of MS colonies using time-lapse cinematography and analyzed the kinetic of their angiogenic potential. As demonstrated by time-lapse studies, APLNR⁺ mesodermal cells placed in semisolid medium possessed a high motility, which was more pronounced before and during the first cell division. Following several divisions, single APLNR⁺ cells formed a core, an immotile structure composed of a small number of tightly packed cells. While APLNR⁺ mesodermal cells lacked endothelial gene expression, molecular profiling of MS colonies at the core stage revealed that these cells acquired angioblastic gene expression profile as indicated by upregulation of FLT1, TEK, CDH5 (VE-cadherin), PECAM1 (CD31), FLI1, SELE (ELAM-1) and ICAM2 endothelial genes. When we collected MS cores (day 3 of clonogenic culture) and placed them on OP9, they formed predominantly VE-cadherin⁺ endothelial clusters, strongly indicating the endothelial nature of the core-forming cells. Subsequently, cells at the periphery of the core underwent endothelial-mesenchymal transition (EndMT) and formed a shell of tightly packed spindle-like cells around the core. When we picked colonies at this stage (day 6 of colony-forming culture) and placed them on OP9, most of the colonies (>70%) grew cell clusters composed of endothelial and mesenchymal cells. In contrast, mature MS colonies collected on day 12 of clonogenic culture formed predominantly clusters of mesenchymal cells, indicating a progressive loss of endothelial potential following colony maturation. Although no CD31 expression was detected in the mesenchymal cells composing mature MS colonies, these cells retained several endothelial traits including surface expression of endothelial tyrosine kinase (TEK or TIE2), FLT1 (VEGFR1) and endomucin. The critical role of EndMT in MS colony formation and MSC development was also congruous with our observation of the suppressive effect of VEGF, a known inhibitor of EndMT,^14,15 on MS colonies. When VEGF was added to MS clonogenic cultures, hESC-derived mesodermal cells were capable of forming angiogenic cores; however, these cores did not transform into mesenchymal cells, indicating that VEGF abrogates MS colony development at the core stage through inhibition of EndoMT. The schematic diagram demonstrating development of mesodermal MSCs is presented in Figure 2.Open in a separate windowFigure 2A model of mesoderm-derived MSC development from hESCs. Coculture with OP9 stromal cells predominantly induces hESC differentiation toward APLNR⁺ mesoderm. APLNR⁺ population contains angiogenic mesodermal precursors with either mesenchymal (mesenchymoangioblast) or hematopoietic (hemangioblast) potentials. Mesenchymoangioblasts and hemangioblasts arise sequentially during differentiation and can be revealed by MS and BL colony formation in response to FGF2. Development of MS and BL colonies in semisolid media proceed through a core stage at which APLNR⁺ cells form clusters of tightly packed cells with angiogenic potential. Subsequently, core-forming cells undergo EndMT giving rise to mesenchymal cells, which form a shell around the core developing into a mature MS colony. VEGF, EndMT inhibitor, blocks MS colony-formation at core stage. The ability of MS-CFCs to generate mesenchymal and endothelial cells can be revealed by coculture of individual colonies with OP9. Similar to MS colonies, BL colonies are formed through establishment of angiogenic core. However, hemangioblast core-forming cells undergo endothelial-hematopoietic transition and grew hematopoietic cells around the core.The close relationship between endothelial and hematopoietic cell development was recognized more than 130 years ago (reviewed by ref. ¹⁶) and confirmed in multiple modern studies.^9,17–22 However, the association between endothelial pre-cursors and MSCs during development was not well established, although cells with endothelial and mural cell potential were identified²³ and the critical role of EndMT in the formation of endocardial cushion²⁴ and testicular cords²⁵ in the embryo was acknowledged. Our hESC-based in vitro studies indicated that formation of mesodermal MSCs proceed through the endothelial stage and likely included at least two successive cycles of cell transitions. Initially APLNR⁺ mesoderm, which consists of fibroblast-like migratory cells, give rise to core structures composed of tightly packed endothelial cells in response to FGF2. Subsequently, endothelial cells forming cores undergo epithelial-mesenchymal transition, i.e., EndMT and form MSCs. The question remains how well this in vitro model reflects in vivo development. Although only sparse data exist regarding MSC precursors in the embryo, development of angiogenic hematopoietic precursors, hemangioblasts was studied more extensively in mammals and birds, and therefore parallels between in vivo and in vitro studies can be drawn. As we demonstrated,⁸ APLNR⁺ mesodermal cells collected from hESCs differentiated on OP9 for 3 days formed disperse BL colonies that identify hemangioblasts in vivo and in vitro.^9,26 Similar to MS colonies, the development of BL colonies required FGF2 and proceeded through angiogenic core formation. However, in contrast to MS cores, BL cores transformed into blood cells, i.e., underwent endothelial-hematopoietic transformation (see Fig. 2). Importantly, in vivo studies identified FGF2 as the essential factor in hemangioblast induction²⁷ analogous to our in vitro observation. In chicken embryo, the activation of FGF signaling leads to aggregation of migrating mesodermal cells adjacent to the endoderm, upregulation of VEGFR2 (KDR) expression, and subsequent formation of angioblasts and hemangioblasts.^28–30 This sequence of events leading to hemangioblast development in vivo considerably resembles what we observed in vitro, and highly suggests accurate recapitulation of embryonic development by our hESC differentiation model. Therefore, searching for an in vivo equivalent of mesenchymonagioblast would be a reasonable next step.In addition to embryonic development, EndMT is also implicated in several pathologies including cancer progression and development of cardiac and renal fibrosis.^31–34 Recently, Olsen group revealed that endothelial cells could be transformed directly into MSCs through overexpression of ALK2 or its activation by TGFβ2 or BMP4,¹⁵ indicating that endothelial cells could be an important source of MSCs in postnatal life. Conversely, the transition from MSCs to endothelial cells, has been also described in reference ³⁵. Based on these observations, a cycle of cell-fate transition from endothelium to MSCs and back to endothelium was proposed as a circuit controlling stem cell state.³⁶ Since multiple parallels could be drawn between EndMT described in adult tissues and during hESC differentiation, one may wonder whether bipotential cells with endothelial and MSC potential similar to embryonic mesenchymoangioblasts are present and constitute an important element of EndMT circuit in adults.In conclusion, the identification of mesenchymoangioblast as a clonogenic precursor of mesoderm-derived MSCs is an important step toward defining pathways of MSC development and specification. In addition, the demonstration of MSC formation from mesoderm through EndMT provides new insights into the mechanisms involved in establishment of MSCs.     

Putting the brakes on cancer cell migration: JAM-A restrains integrin activation

Junctional Adhesion Molecule A (JAM-A) is a member of the Ig superfamily of membrane proteins expressed in platelets, leukocytes, endothelial cells and epithelial cells. We have previously shown that in endothelial cells, JAM-A regulates basic fibroblast growth factor, (FGF-2)-induced angiogenesis via augmenting endothelial cell migration. Recently, we have revealed that in breast cancer cells, downregulation of JAM-A enhances cancer cell migration and invasion. Further, ectopic expression of JAM-A in highly metastatic MDA-MB-231 cells attenuates cell migration, and downregulation of JAM-A in low-metastatic T47D cells enhance migration. Interestingly, JAM-A expression is greatly diminished as breast cancer disease progresses. The molecular mechanism of this function of JAM-A is beyond its well-characterized barrier function at the tight junction. Our results point out that JAM-A differentially regulates migration of endothelial and cancer cells.Key words: JAM-A, integrin, α_vβ₃, FGF-2, breast cancer, cell migration and invasion, T47D, MDA-MB-231, siRNAEndothelial and epithelial cells exhibit cell polarity and have characteristic tight junctions (TJs) that separate apical and basal surfaces. TJs are composed of both transmembrane and cytoplasmic proteins. The three major families of transmembrane proteins include claudins, occludin and JAM family members.^1–3 Additionally, interaction between the peripheral proteins such as PDS-95/Discs large/ZO family (PDZ) domain-containing proteins in TJs plays an important role in maintaining the junctional integrity.^2,4,5JAMs are type I membrane proteins (Fig. 1) predominately expressed in endothelial and epithelial cell TJs, platelets and some leukocytes.^6–8 The classical JAMs are JAM-A, JAM-B and JAM-C, which can all regulate leukocyte-endothelial cell interaction through their ability to undergo heterophilic binding with integrins α_Lβ₂ or α_vβ₃, α₄β₁ and α_Mβ₂ respectively. The cytoplasmic tail of JAMs contains a type II PDZ-domain-binding motif (Fig. 1) that can interact with the PDZ domain containing cytoplasmic molecules such as ZO-1, ASIP/PAR-3 or AF-6.^9,10 Additionally, consistant with their junctional localization and their tendency to be involved in homophilic interactions, JAMs have been shown to modulate paracellular permeability and thus may play an important role in regulating the epithelial and endothelial barrier.^11,12 In addition, ectopic expression of JAM-A in CHO cells promotes localization of ZO-1 and occludin at points of cell contacts, which suggests a role for JAM-A in TJ assembly.^10,13,14 Recently, it has been shown that JAM-A regulates epithelial cell morphology by modulating the activity of small GTPase Rap1 suggesting a role for JAM-A in intracellular signaling.¹⁵Open in a separate windowFigure 1Schematic representation of the domain structure of JAM family proteins. V, variable Ig domain; C2, constant type 2 Ig domain; TM, transmembrane domain; T-II, Type II PDZ-domain binding motif.We have previously shown that JAM-A is a positive regulator of fibroblast growth factor-2 (FGF-2) induced angiogenesis.¹⁶ Evidence was provided to support the notion that JAM-A forms a complex with integrin α_vβ₃ at the cell-cell junction in quiescent human umbilical cord vein endothelial cells (HUVECs) and FGF-2 dissociates this complex.¹⁶ It was further established that inhibition of JAM-A using a function-blocking antibody also inhibits FGF-2 induced HUVECs migration in vitro and angiogenesis in vivo. Overexpression of JAM-A induced a change in HUVECs morphology similar to that observed when treated with FGF-2.¹⁷ Furthermore, overexpression of JAM-A, but not its cytoplasmic domain deletion mutant, augmented cell migration in the absence of FGF-2.¹⁷ In addition, downregulation of JAM-A in HUVECs using specific siRNA, resulted in reduced FGF-2-induced cell migration and inhibition of mitogen activated protein (MAP) kinase activation.¹⁸ These findings clearly suggested that JAM-A positively regulates FGF-2-induced endothelial cell migration. This was further confirmed in vivo by using JAM-A null mouse in which FGF-2 failed to support angiogenesis.¹⁹It is known that JAM-C, a JAM family member, is involved in the process of tumor cell metastasis.²⁰ However, little is known about JAM-A''s role in cancer progression. We recently found that JAM-A is expressed in breast cancer tissues and cell lines.²¹ Based on our studies with endothelial cells it was felt that JAM-A expression in breast cancer cells may also enhance the migratory ability of these cells. Surprisingly, we found an inverse relation between the expression of JAM-A and the metastatic ability of breast cancer cells. T47D cells, which express high levels of JAM-A, are the least migratory; whereas MDA-MB-231 cells, which are highly migratory, are found to express the least amount of JAM-A.²¹ We also found that overexpression of JAM-A in MDA-MB-231 cells caused a change in cell morphology from spindle-like to rounded shape and formed cobblestone-like clusters.²¹ This is consistent with the previous report, that downregulation of JAM-A expression from epithelial cells using siRNA results in the change of epithelial cell morphology.¹⁵ This change in cell morphology by knockdown of JAM-A was attributed to the disruption of epithelial cell barrier function.¹⁵ It was further shown that knockdown of JAM-A affects epithelial cell morphology through reduction of β₁integrin expression due to decreased Rap1 activity.¹⁵ Our observed effect of JAM-A downregulation in T47D cells, however, is not due to downregulation of β₁integrin, since the level of this integrin was not affected in these cells. Interestingly, overexpression of JAM-A significantly affected both the cell migration and invasion of MDA-MB-231 cells. Furthermore, knockdown of JAM-A using siRNA enhanced invasiveness of MDA-MB-231 cells, as well as T47D cells.²¹ The ability of JAM-A to attenuate cell invasion was found to be due to the formation of functional tight junctions as observed by distinct accumulation of JAM-A and ZO-1 at the TJs and increased transepithelial resistance. These results identify, for the first time, a tight junctional cell adhesion protein as a key negative regulator of breast cancer cell migration and invasion.²¹JAM-A has been shown to be important in maintaining TJ integrity.^15,22–25 Disruption of TJs has been implicated to play a role in cancer cell metastasis by inducing epithelial mesenchymal transition.²⁶ Several laboratories, including ours, have shown that cytokines and growth factors redistribute JAM-A from TJs.^16,27,28 Consistent with this finding, it has been shown that hepatocyte growth factor (HGF) disrupts TJs in human breast cancer cells and downregulates expression of several TJ proteins.²⁹ It is therefore conceivable that the loss of JAM-A in highly metastatic cells is a consequence of disruption of TJs. This was further supported by the findings that overexpression of JAM-A forms functional TJs in MDA-MB-231 cells and attenuates their migratory behavior. Our result is the first report correlating an inverse relationship of JAM-A expression in breast cancer cells to their invasive ability.²¹Using cDNA microarray technology, it has been revealed how genes involved in cell-cell adhesion, including those of the TJ, are under or overexpressed in different carcinomas.^15,30 Cell-cell adhesion molecules have been well documented to regulate cancer cell motility and invasion. Of these, the cadherin family have been studied the most.^31,32 It was proposed that a cadherin switch, that is, the loss of E-cadherin and subsequent expression of N-cadherin, may be responsible for breast cancer cell invasion.^33,34 Although the role of cadherins is well-documented, it remains controversial since some breast cancer cell lines that do not express these proteins still posses highly invasive characteristics.^33,34 However, the observed effect of overexpression of JAM-A does not appear to be simply due to the formation of TJs, since individual cells that express increased JAM-A show reduced migration.²¹ This is not surprising, considering the fact that JAM-A in addition to its function of regulating TJ integrity is also shown to participate in intracellular signaling. JAM-A is capable of interacting homotypically as well as heterotypically on the cell surface.^35,36 It has also been shown that it interacts with several cytoplasmic proteins through its PDZ domain-binding motif and recruits signaling proteins at the TJs.³⁷ Recent findings using site-directed mutagenesis suggest that cis-dimerization of JAM-A is necessary for it to carry out its biological functions.³⁸ Our own observations suggest that a JAM-A function-blocking antibody inhibits focal adhesion formation in endothelial cells (unpublished data), whereas overexpresion of JAM-A in MDA-MB-231 cells show increased and stable focal adhesions.²¹ It is therefore conceivable that in quiescent endothelial/epithelial cells JAM-A associates with integrin to form an inactive complex at the TJ (Fig. 2). Growth factors such as FGF-2 signaling dissociates this complex thus allowing dimerization of JAM-A and activation of integrin augmenting cell migration (Fig. 2). On the contrary, in MDA-MB-231 cancer cells, which express low levels of JAM-A and do not form tight junctions, there may not be efficient inactive complex formation between JAM-A and integrin. Overexpression of JAM-A in these cells however, may promote such inactive complex formation leading to inhibition of integrin activation and JAM-A dimerization, both necessary events for cell migration. We are currently in the process of determining the specificity of interaction of JAM-A with integrins. Further experimentation is ongoing to determine the contribution of JAM-A dependent signaling in cell migration.Open in a separate windowFigure 2Schematic representation of JAM-A regulation of cell migration. JAM-A forms an inactive complex with the integrin and sequesters it at the TJs. Growth factor signaling dissociates this complex, promoting integrin activation and JAM-A dimerization leading to cell migration via MAP kinase activation. Ectopic expression of JAM-A in cancer cells may induce its association with integrin, forming an inactive complex and hence attenuation of migration.JAM-A differentially regulates cell migration in endothelial and cancer cells due to its ability to form inactive complex with integrin, making it a metastasis suppressor. The downregulation of JAM-A in carcinoma cells may be detrimental to the survival of breast cancer patients. It is therefore very important to determine the molecular determinants that are responsible for the downregulation of JAM-A during cancer progression. Thus, JAM-A, a molecule that dictates breast cancer cell invasion, could be used as a prognostic marker for metastatic breast cancer.     

Trading mtDNA uncovers its role in metastasis

It has been controversial for many years of whether mtDNA mutations are involved in phenotypes related to cancer due to the difficulty in excluding possible involvement of nuclear DNA mutations in these phenotypes. We addressed this issue by complete trading of mtDNAs between tumor cells expressing different metastatic phenotypes. Resultant trans-mitochondrial cybrids share the same nuclear background, but possess mtDNA from tumor cells expressing different metastatic phenotypes, and thus can be used to uncover the role of mtDNA in these phenotypes. The results showed that mtDNA controls development of metastasis in tumor cells, while tumor development is controlled by nuclear genome.Key words: pathogenic mtDNA mutations, respiration defects, enhanced glycolysis, ROS overproduction, rho-zero cells, mtDNA transfer technology, metastasisHuman mtDNAs with pathogenic mutations inducing significant respiration defects have been shown to be closely associated with mitochondrial diseases.^1,2 Although mitochondrial respiratory function is controlled by both nuclear and mitochondrial genomes, the pathogenicity of these mtDNA mutations has been proven by co-transmission of the mutant mtDNAs and mitochondrial respiration defects to mtDNA-less (ρ⁰) human cells: the resultant trans-mitochondrial cybrids sharing the same nuclear backgrouond showed respiration defects, only when they accumulated the mutated mtDNA from the patients.^3–6 Moreover, we generated transmitochondrial mito-mice sharing the same nuclear background, but carrying various proportions of mtDNA with a pathogenic mutation, and provided model systems for studying exactly how mtDNAs with pathogenic mutations are transmitted and distributed in tissues resulting in the pathogenesis of mitochondrial diseases that show various clinical phenotypes.^7–9With respect to the involvement of mtDNA in tumor phenotypes, it has been proposed that most chemical carcinogens bind preferentially to mtDNA rather than to nuclear DNA in mammalian cells,^10–12 and thus, mtDNA should be the major cellular target of chemical carcinogens, and resultant creation of mutations in mtDNAs is responsible for expression of tumor phenotypes.¹²Although, there has been no direct evidence for creation of mtDNA mutations by chemical carcinogens, and for their contribution to tumor development in mammalian cells, recent studies showed that somatic mtDNA mutations accumulated in human colorectal tumors¹³ and in various tumor types¹⁴ rather than in normal cells of the same subjects, probably by the clonal expansion of the mutated mtDNAs along with the repeated division of tumor cells. Many subsequent studies supported preferential accumulation of mutated mtDNAs in tumor cells,^15–18 suggesting that mutated mtDNAs in tumor cells have acquired replication advantages to be homoplasmic. However, these studies did not address the fundamental question of whether the mutated mtDNAs are involved in tumor development.Our previous studies directly addressed this issue using transmitochondrial cybrids obtained by mtDNA trading between normal and tumor cells, and provided convincing evidence that mutations in nuclear DNA, but not in mtDNA were involved in tumor development in the mouse^19,20 and in human cultured cells.^21,22 The possibility that these observations may represent some specific tumor cases can be excluded since there has been no statistical evidence for association of tumor development and pathogenic mtDNA mutations in the patients with mitochondrial diseases expressing respiration defects caused by pathogenic mutations in mtDNA. The possibility that some polymorphic mtDNA mutations that do not induce respiration defects, but somehow contribute to tumor development also can be excluded, because there has been no statistical evidence for the presence of maternal inheritance of tumor development in spite of the strictly maternal inheritance of mammalian mtDNA.^23,24Nonetheless, it was still possible that mtDNA mutations are involved in other processes than oncogenic transformation of normal cells to develop tumors, such as in malignant progression of tumor cells to develop a metastatic potential. Recent studies demonstrated that mitochondrial respiration defects in TCA-cycle enzymes caused by nuclear DNA mutations controls tumor phenotypes as a consequence of induction of a pseudo-hypoxic pathway under normoxia.^25–27 Thus, some mtDNA mutations also induce the pseudo-hypoxic pathway under normoxia by inducing mitochondrial respiration defects. However, there has been no direct evidence for involvement of mtDNA mutations in malignant progression or in the regulation of the pseudo-hypoxic pathway under normoxia, because of the difficulty in excluding possible contribution of nuclear DNA mutations in these processes.²⁸Recently, we addressed this issue using trans-mitochondrial cybrids²⁹ obtained by complete trading of mtDNAs between highly and poorly metastatic mouse lung carcinoma cells (Fig. 1). By this approach, we could provide convincing evidence for the control of malignant progression of tumor cells to develop metastatic potential by mtDNA:²⁹ all the trans-mitochondrial cybrids with mtDNA from highly metastatic tumor cells expressed high metastatic potential, while those with mtDNA from poorly metastatic tumor cells expressed low metastatic potential, irrespective of whether their nuclear genome was derived from highly or poorly metastatic tumor cells. The findings in our study²⁹ can be summarized as follows: (1) A missense G13997A mutation in the ND6 gene of mtDNA from highly metastatic lung tumor cells induces a complex I defect, and reversibly controls malignant progression of tumor cells to develop metastatic potential, but does not control oncogenic transformation of normal cells to develop tumors; (2) The complex I defect simultaneously induces enhanced glycolysis and ROS overproduction, but induction of metastasis is due to ROS overproduction; (3) ROS overproduction induces metastasis not by acceleration of genetic instability as usually proposed, but by reversible upregulation of nuclear-coded genes related to metastasis, such as Mcl-1; (4) ROS scavengers are therapeutically effective in suppressing mtDNA-mediated metastasis.Open in a separate windowFigure 1Scheme for the isolation of the trans-mitochondrial cybrids with completely exchanged mtDNA between parental cells expressing different metastatic phenotypes. Trading mtDNA shown in this scheme uncovered a role of mtDNA in metastasis. For trading mtDNA, parental P29 and A11 cells were treated with ditercalinium, an antitumor bis-intercalating agent, to isolate ρ⁰P29 and ρ⁰A11 cells (*), which have no mtDNA. Complete depletion of mtDNA was confirmed by PCR analysis. Enucleated cells of the mtDNA donors were prepared by their pretreatment with cytochalasin B and centrifugation. Resultant cytoplasts were fused with ρ⁰ cells by polyethylene glycol to obtain trans-mitochondrial cybrids. High metastatic potential is transferred to the P29mtA11 cybrids with the transfer of mtDNA from the A11 cells, and poor metastatic potential is transferred to the A11mtP29 cybrids with the transfer of mtDNA from the P29 cells. Involvement of cytoplasmic factors other than mtDNA from the A11 cells in expression of the high metastatic potential in the P29mtA11 cybrids can be ruled out by the observations that the A11mtP29 cybrids lost their high metastatic potential, even though they always contain cytoplasmic factors transcribed by the nuclear genes derived from the A11 cells.Thus, our study partly resolves the controversial issue on the relevance or irrelevance of mtDNA mutations in tumor development and/or tumor phenotypes by showing that mutations in mtDNA control development of metastasis in tumor cells.²⁹ Considering that complex I defects simultaneously induce enhanced glycolysis under normoxia (the Warburg effect) and ROS overproduction,²⁹ it remains possible that the Warburg effect alone can control metastasis independently from ROS overproduction. More recently, we examined this possibility by generating trans-mitochondrial cybrids with the deletion mutant mtDNA, which can be expected to induce overall respiration defects, and express enhanced glycolysis under normoxia, but not express ROS overproduction. The results showed that the Warburg effect alone did not control metastasis.     

Rap GTPase-mediated adhesion and migration: A target for limiting the dissemination of B-cell lymphomas?

B-cell lymphomas, which arise in lymphoid organs, can spread rapidly via the circulatory system and form solid tumors within multiple organs. Rate-limiting steps in this metastatic process may be the adhesion of lymphoma cells to vascular endothelial cells, their exit from the vasculature and their migration to tissue sites that will support tumor growth. Thus proteins that control B-cell adhesion and migration are likely to be key factors in lymphoma dissemination, and hence potential targets for therapeutic intervention. The Rap GTPases are master regulators of integrin activation, cell motility and the underlying cytoskeletal, adhesion and membrane dynamics. We have recently shown that Rap activation is critical for B-lymphoma cells to undergo transendothelial migration in vitro and in vivo. As a consequence, suppressing Rap activation impairs the ability of intravenously injected B-lymphoma cells to form solid tumors in the liver and other organs. We discuss this work in the context of targeting Rap, its downstream effectors, or other regulators of B-cell adhesion and migration as an approach for limiting the dissemination of B-lymphoma cells and the development of secondary tumors.Key words: B-cell lymphomas, Rap GTPases, extravasation, chemokines, integrins, metastasisB-cell lymphomas are frequently occurring malignancies that are often aggressive and difficult to treat. Abnormally proliferating B cells that acquire survival-promoting mutations originate within the bone marrow or the lymphoid organs but can traffic via the blood and lymphatic systems to other organs, where they can form solid tumors. A consequence of the genetic mechanisms that generate a large repertoire of antigen-detecting B-cell receptors (BCR) and antibodies is an increased frequency of chromosomal translocations and mutations that can lead to oncogenic transformation.¹ During B-cell development in the bone marrow, the vast diversity of the BCR repertoire within an individual is generated by the random rearrangement of the VDJ gene segments that encode the BCR. Subsequent to antigen binding, highly proliferating B cells within the germinal centers of secondary lymphoid organs undergo somatic hypermutation of the genes encoding the immunoglobulin portion of the BCR in order to generate antibodies of higher affinity (“affinity maturation”). These cells can also undergo a second DNA rearrangement event associated with immunoglobulin class switching. Aberrant DNA rearrangements or somatic hypermutation can lead to oncogenic transformation. As examples, translocation of the c-myc gene into the IgH locus is characteristic of Burkitt''s lymphoma whereas somatic hypermutation of genes that encode prosurvival proteins (e.g., pim-1) is associated with diffuse large B-cell lymphomas,² the most common type of non-Hodgkin lymphoma.The ability of B-cell lymphomas to spread to multiple organs reflects the migratory capacity of their normal counterparts. B cells circulate continuously throughout the body via the blood and lymphatic systems. The extravasation of B cells out of the blood and into tissues is a multi-step process that requires selectin-mediated rolling on the surface of vascular endothelial cells, intergin-mediated firm adhesion to the endothelial cells, and migration across the endothelial cell monolayer that makes up the vessel wall (Fig. 1).^3–6 These steps are orchestrated by chemokines and adhesion molecules that are displayed on the surface of the vascular endothelial cells. Chemokines initiate signaling within the B cell that results in integrin activation. The collaboration between chemokine receptor signaling and outside-in integrin signaling causes B cells to reorganize their cytoskeleton. This cytoskeletal reorganization allows B cells to spread on the surface of the vascular endothelial cells, migrate to sites suitable for extravasation (e.g., junctions between endothelial cells) and then deform themselves in order to move across the endothelial cell layer.⁷ The ability of B-cell lymphomas to follow these constitutive organ-homing cues allows them to spread to multiple organs throughout the body, making them difficult to combat. Diffuse large B-cell lymphomas are highly aggressive precisely for this reason and readily spread to the liver, kidneys and lungs.⁸ Thus, identifying key proteins that regulate the extravasation of B-cell lymphomas could suggest new therapeutic strategies for treating these malignancies.⁹Open in a separate windowFigure 1Rap activation is required for multiple steps in lymphoma dissemination. B-cell lymphomas exit the vasculature using the same mechanisms as normal B cells. Once B cells are tethered via selectin-mediated rolling, chemokines immobilized on the surface of vascular endothelial cells convert integrins to a high affinity state via a mechanism that involves activation of the Rap GTPases. This permits firm adhesion. Adhered B cells migrate across the endothelium and then send out actin-rich protrusions, which penetrate the endothelial barrier to reach the subendothelial matrix. The formation of these membrane processes, and the subsequent movement of the cells through the junctions, requires activation of the Rap, Rho and Rac GTPases. Once in the tissue, B-lymphoma cells assume a polarized morphology and can migrate towards optimal growth niches.The ubiquitously-expressed Rap GTPases are master regulators of cell adhesion, cell polarity, cytoskeletal dynamics and cell motility.¹⁰ Receptor-induced conversion of the Rap GTPases to their active GTP-bound state (Rap-GTP) allows them to bind multiple effector proteins and thereby orchestrate their localization and function. These downstream effectors of Rap-GTP control integrin activation, actin polymerization and dynamics and the formation of protrusive leading edges in migrating cells (see below and Fig. 2). In both normal B cells and B-lymphoma cell lines, signaling via chemoattractant receptors, the BCR and integrins all activate Rap.^11–13 Moreover, we have shown that chemokine-induced Rap activation is essential for the chemoattractants CXCL12 (SDF-1), CXCL13 and sphingosine-1-phosphate (S1P) receptors to stimulate B-cell migration and adhesion.^12,14 Rap activation is also important for receptor-induced actin polymerization, cell spreading and cytoskeletal reorganization in both primary B cells and B-lymphoma cells.¹⁵ These findings suggested that Rap activation might be essential for the in vivo metastatic spread of B-cell lymphomas.Open in a separate windowFigure 2The Rap GTPases are master regulators of actin dynamics, cell morphology, cell polarity and integrin-mediated adhesion. The Rap GTPases are activated subsequent to the binding of chemokines to their receptors or activated integrins to their ligands. The active GTP-bound form of Rap binds effector proteins that promote integrin activation, actin polymerization and membrane protrusion, as well as activation of the Pyk2 and FAK tyrosine kinases, which modulate cell spreading, adhesion and migration. Rap-GTP also plays a key role in establishing cell polarity and may direct membrane vesicles to the leading edge of the cell. See text for details. MTOC, microtubule-organizing center.To test this hypothesis, we suppressed Rap activation in A20 murine B-lymphoma cells, a cell line derived from an aggressive diffuse large B-cell lymphoma. We blocked Rap activation in these cells by expressing a Rap-specific GTPase-activating protein (GAP), RapGAPII, which enzymatically converts Rap1 and Rap2 proteins to their inactive GDP-bound states. Injecting stable A20/RapGAPII and A20/empty vector transfectants intravenously into mice showed that Rap activation was required for these cells to form solid lymphomas within organs such as the liver.¹⁶ Solid tumor formation was delayed and reduced when A20/RapGAPII cells were injected instead of A20/control cells. Strikingly, the lymphoma cells isolated from the tumors that developed in mice injected with A20/RapGAPII cells had downregulated RapGAPII expression and regained the ability to activate Rap. Thus tumor formation reflected a strong in vivo selection for lymphoma cells capable of activating Rap. This indicates that Rap-dependent signaling is critical for the metastatic spread of B-cell lymphomas.The ability of B-lymphoma cells to exit the vasculature and migrate into the underlying tissue is likely to be a rate-limiting step in the metastasis of B-cell lymphomas. We showed that this extravasation step is a Rap-dependent process for B-cell lymphomas. To do this, we performed competitive in vivo homing assays in which differentially-labeled A20/vector and A20/RapGAPII cells were co-injected into the tail veins of mice.¹⁶ Analyses performed 1–3 days after injecting the cells showed that A20/RapGAPII cells exhibited a greatly reduced ability to arrest and lodge in the liver, compared to control cells. The liver produces large amounts of the chemokine CXCL12 and is a major site of lymphoma homing and tumor formation. More detailed studies revealed that the control A20 cells that lodged in the liver had entered the liver parenchyma and had an elongated morphology, as expected for cells that are migrating within the tissue and interacting with resident cells. In contrast, a larger fraction of the A20/RapGAPII cells were round and appeared to still be within the vasculature. These findings suggest that Rap activation is required for efficient extravasation of lymphoma cells in vivo, as had previously been shown for in T cells in vitro.¹⁷Leukocyte extravasation is a multi-step process that requires initial adhesion to the vascular endothelium followed by crawling on the luminal surface of the endothelial cells until a suitable site for migration through the endothelial barrier is located. We found that Rap activation was required for the initial adhesion of A20 cells to vascular endothelial cells in vitro.¹⁶ Whether integrin-mediated adhesion is an absolute requirement for tumor cells to arrest within organ vasculature remains an open question as tumor cells can be physically trapped in small vessels in a manner that is independent of integrins or other adhesion molecules (Freeman SA, unpublished data). In contrast, the ability of lymphoma cells to generate polarized membrane protrusions that invade junctions between vascular endothelial cells and then move through the junctions is likely to have a strong dependence on Rap-mediated integrin activation and Rap-mediated cell polarization and cytoskeletal reorganization. Indeed, we found that Rap activation was required for A20 B-lymphoma cells to form membrane projections that penetrated endothelial junctions in vitro, and for the subsequent transendothelial migration of A20 cells.¹⁶In addition to this well-characterized paracellular mode of extravasation in which leukocytes crawl across endothelial cells until they arrive at cell-cell junctions and then migrate across the endothelial cell layer, leukocytes can also extravasate via a transcellular route.¹⁸ T cells can send invadopodia through endothelial cells, which upon contacting the subendothelial matrix pull the cell through and across the endothelial cell. The paracellular and transcellular routes of leukocyte extravasation may involve distinct modes of leukocyte motility and cytoskeletal reorganization. For example, activation of WASp and Src is required for transcellular extravasation of T cells, but dispensable for paracellular extravasation.¹⁸ Our data suggest that Rap activation is involved in the paracellular extravasation of B-cell lymphomas. It is not known if lymphoma cells, which are considerably larger than normal leukocytes, can undergo transcellular extravasation, and if so, whether Rap-dependent signaling is required. Determining the relative contributions of these two modes of extravasation, as well as their underlying molecular mechanisms, could facilitate the development of therapeutic approaches for reducing lymphoma cell extravasation and dissemination.Rap GTPases are ubiquitously expressed and are involved in critical processes such as the formation of tight junctions between vascular endothelial cells.¹⁹ Therefore, targeting downstream effectors of Rap that mediate specific aspects of adhesion and migration may be a more reasonable way to limit lymphoma dissemination than targeting Rap activation. As shown in Figure 2 and reviewed by Bos,¹⁰ the effector proteins that are regulated either directly or indirectly by Rap-GTP control several modules that are critical for cell adhesion and migration.Activated Rap is an essential component of the inside-out signaling pathway by which chemokine receptors activate integrins. Rap-GTP recruits the adaptor protein RapL as well as RIAM/talin complexes to the cytoplasmic domains of integrins.^20,21 This results in conformational changes in the integrin extracellular domains that increase their affinity for adhesion molecules, such as those present on the surface of vascular endothelial cells. Actin-dependent intracellular forces exerted by talin on the integrin cytoplasmic domains also increase integrin affinity²² and may be regulated by Rap-GTP, which promotes actin polymerization (see below).Effector proteins that bind Rap-GTP include upstream activators of Rac and Cdc42,^23,24 GTPases that promote dynamic actin polymerization at the leading edge of migrating cells and at the growing ends of membrane protrusions. Activated Rac and Cdc42 act via the WASp and WAVE proteins to induce branching actin polymerization that drives membrane protrusion and the formation of lamellipodia and filopodia. Other Rap effectors, the RIAM²⁵ and AF-6 adaptor proteins,²⁶ allow Rap-GTP to recruit Ena/Vasp and profilin, proteins that prime actin monomers for incorporation into actin filaments, a rate-limiting step in actin filament assembly.The Pyk2 and FAK tyrosine kinases are key regulators of cell adhesion, cell migration and cell morphology, and we have shown that they are also downstream targets of Rap-GTP signaling.²⁷ Rap-dependent actin dynamics is critical for the activation of Pyk2 and FAK in B-lymphoma cells. Moreover the kinase activities of Pyk2 and FAK are required for B cell spreading, a key aspect of cell adhesion and motility.²⁷ The importance of this Rap/Pyk2 signaling module is supported by the observation that B cells from Pyk2-deficient mice have a severe defect in chemokine-induced migration.²⁸Rap effectors also promote the establishment of cell polarity, another key aspect of cell motility. Rap-GTP binds the evolutionarily-conserved Par3/6 polarity complex²⁹ and promotes the microtubule-dependent transport of vesicles containing integrins to the leading edge of migrating cells and to cell-cell contact sites such as immune synapses.^30,31A key question is whether modulating the expression or activity of individual targets of Rap signaling can effectively limit the dissemination of B-cell lymphomas. An exciting recent paper supports the idea that targeting proteins involved in cell motility may be an effective way to limit the spread and growth of B-cell lymphomas.⁹ Using a library of short hairpin RNAs (shRNAs) directed against 1,000 genes thought to be involved in lymphoma progression, Meachem et al. found that two regulators of the actin cytoskeleton, Rac2 and twinfilin (Twf1), were key determinants of lymphoma motility, invasiveness and progression. shRNA-mediated knockdown of either Rac2 or Twf1 expression dramatically inhibited the growth of Eµ-myc B-cell lymphomas in mice, a model for the development of human Burkitt lymphomas. The decreased lymphoma tumorgenicity, as well as the decreased ability of the lymphoma cells to engraft in the spleen and bone marrow and then metastasize to secondary sites such as the liver was associated with the cells'' inability to migrate and crawl in vitro. This is consistent with our finding that inhibiting the in vitro migration and adhesion of B-lymphoma cells by suppressing Rap activation correlated with reduced extravasation and tumor formation in vivo.The involvement of both Rap and Rac2 in lymphoma motility and dissemination may reflect the fact that these two GTPases lie in the same pathway. Rap-GTP has been shown to bind the Rac activator Vav2 and promote Rac activation.²³ Conversely, Batista and colleagues showed that Rac2 acts upstream of Rap to promote Rap activation and modulate B-cell adhesion and immune synapse formation.³² Although the interrelationship of Rap and Rac2 in B-cell lymphomas remains to be clarified, the Rac2/Rap signaling module is a potential target for limiting the spread of B-cell lymphomas. Inhibiting this Rac2/Rap module that controls B-cell motility and adhesion may reduce both the extravasation of lymphoma cells into organs as well as the ability of B-lymphoma cells to crawl to sites within the organ where they can establish a suitable metastatic niche. Migration through the subendothelial stroma to find optimal growth niches is a rate-limiting step in the dissemination of many types of tumors.³³ Blocking Rap-dependent adhesion may also prevent B-lymphoma cells from forming critical adhesive interactions with tissue-resident stromal cells. In vitro, the survival of many B-cell lymphomas depends on integrin engagement^34,35 and the subsequent activation of pro-survival signaling pathways (e.g., the PI 3-kinase/Akt pathway) by integrin signaling.³⁶ It is not known whether Rap-dependent adhesion and the ensuing integrin-mediated survival signaling are required for B-cell lymphomas to form solid tumors at secondary sites in vivo.A series of recent papers has identified the hematopoietic lineage-restricted adaptor protein kindlin-3 as a key regulator of integrin activation in leukocytes. Kindlin-3 is required for leukocyte adhesion in vitro and for in vivo extravasation,^37–39 making it a potential target for limiting the spread of B-cell lymphomas. Kindlin-3 binds to the cytoplasmic domain of several integrin beta subunits but the mechanism by which it promotes integrin activation is not known. An interesting question is whether Rap-GTP, or the RapL/RIAM/talin complexes that are recruited to integrins by Rap-GTP, regulate the localization or function of kindlin-3. Whether or not Rap and kindlin-3 act in the same pathway, it would be interesting to test whether knocking down the expression of kindlin-3 reduces the dissemination of B-cell lymphomas in either the A20 cell model we have used or the Eµ-myc B-cell lymphoma model used by Meachem et al.⁹Although we have thus far referred to the Rap GTPases collectively as “Rap,” there are five Rap GTPases in humans and mice, Rap1a, Rap1b, Rap2a, Rap2b and Rap2c, each encoded by a separate gene. Several reports have suggested distinct functions for Rap1 versus Rap2,^14,40 but it is not known to what extent the functions of the five Rap proteins are redundant or unique. Although many studies have not assessed Rap2 activation, loss-of-function approaches such as overexpressing Rap-specific GAPs or expressing the dominant-negative Rap1N17 protein may suppress the activation of all Rap proteins. Nevertheless, the possibility that different Rap proteins have distinct functions, coupled with cell type-specific differences in the expression of the Rap proteins, may present additional opportunities for targeting Rap signaling in tumor cells. Rap1b is much more abundant than Rap1a in B cells and recent work has shown that Rap1b-deficient murine B cells exhibit impaired migration and adhesion in vitro, as well as impaired in vivo homing.^41,42 If B-lymphoma cells also express much more Rap1b than Rap1a, then Rap1b could be a target for limiting the spread of these malignant B cells. An important caveat is that Rap1b is also the most abundant Rap1 isoform in platelets and plays a critical role in platelet aggregation and clotting.^43,44As master regulators of cell adhesion and migration, the Rap GTPases and the signaling pathways they control are obvious therapeutic targets for limiting the spread of B-cell lymphomas. Other signaling pathways that impact B-cell migration and adhesion, perhaps independently of Rap, are also attractive targets. Our in vivo experiments and those of Meachem et al.⁹ provide direct evidence that interfering with key regulators of adhesion and migration can dramatically limit the dissemination of B-cell lymphomas and the development of secondary tumors in critical organs. Further studies are needed to determine if this approach would be a useful therapeutic strategy for patients with B-cell lymphoma.Finally, it will be of interest to determine whether gain-of-function mutations that increase Rap signaling, or activate other pathways that promote B cell migration and adhesion, contribute to the aggressiveness of certain types of B-cell lymphomas. Increased Rap activation is associated with enhanced invasiveness in several types of tumors.^45,46 If this were true for B-cell lymphomas, then Rap-GTP levels could be a useful prognostic marker for aggressive lymphomas, in addition to being a potential therapeutic target.     

L1 cell adhesion molecules as regulators of tumor cell invasiveness

Fast growing malignant cancers represent a major therapeutic challenge. Basic cancer research has concentrated efforts to determine the mechanisms underlying cancer initiation and progression and reveal candidate targets for future therapeutic treatment of cancer patients. With known roles in fundamental processes required for proper development and function of the nervous system, L1-CAMs have been recently identified as key players in cancer biology. In particular L1 has been implicated in cancer invasiveness and metastasis, and has been pursued as a powerful prognostic factor, indicating poor outcome for patients. Interestingly, L1 has been shown to be important for the survival of cancer stem cells, which are thought to be the source of cancer recurrence. The newly recognized roles for L1CAMs in cancer prompt a search for alternative therapeutic approaches. Despite the promising advances in cancer basic research, a better understanding of the molecular mechanisms dictating L1-mediated signaling is needed for the development of effective therapeutic treatment for cancer patients.Key words: L1CAMs, cancer, metastasis, axon guidance, cancer stem cell, migration, invasionA major obstacle in oncology is the early diagnosis and curative therapeutic intervention of locally invasive cancers that rapidly disseminate from the primary tumor to form metastases. The standard treatment for malignant tumors consists of surgical removal of the tumor mass followed by chemo- and radiotherapy in order to eradicate the remaining cancer cells. Despite such aggressive intervention, a population of resistant cancer cells often remains intact and is thought to be the source of cancer recurrence.During the past decades, cancer basic research has focused on determining the molecular mechanisms underlying cancer initiation and progression that can provide a basis for the development of new and effective therapeutic treatments for cancer patients. An important finding was the discovery that cancer onset and development are often associated with alterations in the expression of cell adhesion molecules, which are likely to stimulate tumor cell invasiveness by signaling mechanisms that enhance cell migration.¹ The L1 family of neural cell adhesion molecules (L1-CAMs), which is comprised of four structurally related transmembrane proteins L1, CHL1, NrCAM and neurofascin (Fig. 1), is now in the spotlight of cancer research due to their upregulation in certain human tumors. L1-CAMs are transmembrane molecules of the immunoglobulin superfamily, characterized by an extracellular region of six immunoglobulin-like domains and four to five fibronectin type III repeats, followed by a highly conserved cytoplasmic domain, which is reversibly linked to the cell cytoskeleton through binding to ankyrin and ERM proteins (ezrin-radixin-moesin).² Its multi-domain structure allows complex heterophilic interactions with diverse cell receptors, although homophilic interactions also have a crucial role in L1-CAMs mediated signaling.Open in a separate windowFigure 1L1-CAMs: All have 6 Ig domains and 4–5 FN domains. The 186 kD Neurofascin isoform has a mucin-like Pro/Ala/Thr-rich (PAT) domain, while the 155 kD has only the 4 FN domains. RGD and DGEA motifs interact with integrins, while the FigQ/AY motif binds to ankyrin. ERM binding sites are indicated. The RSLE motif in L1 recruits AP2/clathrin adaptor for endocytosis.A wealth of studies has revealed L1-CAMs as pivotal components for proper development of the nervous system through regulation of cell-cell interactions. L1-CAMs have critical roles in neuronal migration and survival, axon outgrowth and fasciculation, synaptic plasticity and regeneration after trauma.² Neither CHL1 nor L1 is present on mature astrocytes, oligodendroglia or endothelial blood vessel cells in the brain, but CHL1 is upregulated in astrocytes upon injury³ and is present on oligodendroglial precursors.^4,5 During neural development, L1 plays an important role in the migration of dopaminergic neuronal cell groups in the mesencephalon and diencephalon.⁶ In the cerebellum, L1 is required for the inward migration of granule neurons from the external granular layer and cooperates with NrCAM in regulating neuronal positioning.² Similarly, CHL1 controls area-specific migration and positioning of deep layer cortical neurons in the neocortex.⁷ In addition to its role in neuronal precursor positioning, L1 plays a crucial role in axon guidance, which is governed by repellent and attractive response mechanisms directed by Ephrins and Semaphorins and their receptors (Ephs, Neuropilins, Plexins).² The importance of L1-CAMs in the development and function of the nervous system is exemplified by developmental neuropsychiatric disorders that are associated with mutation or genetic polymorphisms in genes encoding L1 (X-linked mental retardation) and CHL1 (low IQ, speech and motor delay). Polymorphisms in L1 and CHL1 genes are also associated with schizophrenia, and NrCAM gene polymorphisms are linked to autism in some populations.²Recent studies have described upregulation of L1 in a variety of tumor types. Overexpression of L1 correlates with tumor progression and metastasis in certain human gliomas,⁸ melanoma,⁹ ovarian¹⁰ and colon carcinomas.^11–13 Interestingly, L1 was found to be present only in cells at the invasive front of colon cancers but not in the tumor mass.¹² L1 is also associated with micrometastasis to both lymph nodes and bone marrow in patients bearing other cancers, suggesting a potential role in early metastatic spread.¹¹ L1 has now been pursued as both a biomarker and a powerful prognostic factor, indicative of poor outcome for patients as observed for epithelial ovarian carcinoma¹⁰ and colorectal cancer.¹¹ More recently, L1 has been shown to be overexpressed in a small fraction of glioma cells, termed glioma stem cells, which are capable of self-renewal and generate the diverse cells that comprise the tumor.¹⁴ First characterized in acute myeloid leukemia,¹⁵ cancer stem cells have been recently described in a variety of solid tumors, including breast cancer, lung cancer and gastrointestinal tumors.¹⁶ In gliomas, L1 expression was shown to be required for maintaining the growth and survival of glioma stem cells.¹⁴ These findings suggest that L1 may be implicated not only in cancer invasiveness but also in cancer survival. It will be important to determine if L1 is also upregulated in other cancer stem cells as well as to define the role of L1-mediated signaling in other cancers. Although not extensively investigated, NrCAM has also been shown to be overexpressed in glioblastoma cell lines and several cases of high grade astrocytoma¹⁷ and ependymomas.¹⁸ Studies are needed to address whether CHL1 and neurofascin play analogous roles in cancer onset and progression.The molecular mechanisms of L1-mediated signaling that govern the migration of neuronal precursors and guidance of axons during the development of the nervous system may also be used by cancer cells to facilitate invasion and cancer progression. Integrins are well-characterized cooperative partners for L1-CAMs, and signal transduction pathways activated by this complex are known to promote cell adhesion and directional motility. L1/integrin-mediated signaling may converge with growth factor signaling networks to promote motility. Like L1, CHL1 cooperates with integrins to stimulate migration. All L1-CAMs reversibly engage the actin cytoskeleton through a conserved motif FigQ/AY in the cytoplasmic domain that contains a crucial tyrosine residue required for binding the spectrin adaptor ankyrin. Phosphorylation of the FigQY tyrosine decreases ankyrin binding, whereas dephosphorylation promotes L1-ankyrin interaction. Dynamic adhesive interactions controlled by phosphorylation/dephosphorylation of the ankyrin motif in L1 family members may enable a cell to cyclically attach and detach from the ECM substrate or from neighboring cells, thus facilitating migration.¹ Another way L1 promotes cell migration is by stimulating endocytosis of integrins, reducing cell adhesion to the extracellular matrix.¹⁹ Thus, it is reasonable to speculate that upregulation of L1 in cancer may result in increased L1-mediated signaling and, consequently, increased cell migration.L1-CAMs are cleaved by metalloproteases, releasing functionally active ectodomain fragments that are laid down as “tracks” on the extracellular matrix (ECM). These fragments can cause autocrine activation of signal transduction pathways, promoting cell migration through heterophilic binding to integrins.²⁰ Specifically, L1 is cleaved constitutively or inducibly by the ADAM family metalloproteases (a disintegrin and metalloprotease) ADAM10 and ADAM17, which stimulates cell migration and neurite outgrowth during brain development.^20,21 In colon cancer, L1 colocalizes with ADAM 10 at the invasive front of the tumor tissue, suggesting that L1 shedding may play a role in cancer invasiveness.¹² Similarly, CHL1 is shed by ADAM8, which was reported to promote cell migration and invasive activity of glioma cells in vitro and is highly expressed in human brain tumors including glioblastoma multiforme, correlating with invasiveness in vivo.²² Furthermore, NrCAM, found in pancreatic, renal and colon cancers, is subject to ectodomain shedding,²³ but its function in regulating cell migration or invasion has not yet been studied.Given the newly recognized roles of L1 in tumor progression, a growing body of experimental studies has explored novel therapeutic approaches targeting L1-CAMs. Antibody-based therapeutic strategies are being pursued to functionally inhibit homophilic and heterophilic interactions of cell adhesion molecules to suppress tumor invasive motility. L1 monoclonal antibodies reduce in vivo growth of human ovarian and colon carcinoma cells in mouse xenograft models.^13,24,25 L1 targeting using lentiviral-mediated short hairpin RNA (shRNA) interference decreases growth and survival of glioma stem cells in vitro, suppresses tumor growth, and increases survival of tumor-bearing animals.¹⁴ These findings raise the possibility that L1 represents a cancer stem cell-specific therapeutic target for improving the treatment of malignant gliomas and other brain tumors. Cancer stem cells represent a potential target for future treatment of different cancer as these cells are believed to be responsible for cancer recurrence.²⁶ Promoting cancer stem cell differentiation by drug treatment could potentially reduce stem cells properties of self-renewal and proliferation, leading to inhibition of tumor growth.Inhibitors of metalloproteases that block L1-CAM shedding represent a potentially novel approach to curtailing tumor invasiveness. Chemical inhibitors of ADAMS are appealing for glioma therapy due to their diffusability, which circumvents blood-brain barrier limitations. Another novel approach involves the secreted axon repellent protein, Semaphorin 3A (Sema3A). L1-CAMs serve as co-receptors for Sema3A by cis binding in the plasma membrane to Neuropilin-1, important for repellent axon guidance.² Interestingly, Sema3A inhibits invasiveness of prostate cancer cells²⁷ and migration and spreading of breast cancer cells in in vitro assays,²⁸ and thus may also be mediated by L1-CAMs. Such an approach could be potentially useful in mitigating invasion of cancer cells in gliomas and other tumors that are known to express L1 and Neuropilins. However, effective strategies for some types of cancer can promote cancer progression in other types. For example, Sema3A has been shown to contribute to the progression of pancreatic cancer²⁹ and colon cancer.³⁰ Thus, it is imperative that the molecular mechanisms underlying L1-mediated signaling are understood in a tissue specific manner. Despite the promising advances in cancer basic research, much more research is needed to better design strategies for cancer therapy.     

Environmental stimuli and intestinal stem cell behavior

Comment on: Wong VWY, et al. Nat Cell Biol 2012; 14:401-8.The intestine carries out important functions related to digestion and absorption. It is composed of three distinct layers, an outer muscle layer, a mesenchymal layer and the epithelial layer. The epithelial layer forms the protective barrier that faces the luminal content of the intestine. In order to maintain barrier function the epithelial layer needs constant replenishment. This is ensured by continuous cellular replication in proliferative crypt compartments. Following exit from the crypt, cells adopt fates along either secretory or absorptive lineage and will, after three to four days, be exfoliated into the lumen of the intestine from the tips of the villi. Intestinal stem cells located at the bottom of the proliferative crypt compartment ensure lifelong maintenance of the organ (Fig. 1A).Open in a separate windowFigure 1. Diagram of the intestinal stem cell niche. (A) Lgr5-expressing columnar-based crypt cells (CBCs) intercalated between Paneth cells are indicated in green. Stem cells located in position +4 are yellow. Lrig1 is expressed in a gradient along the niche axis with highest expression in the CBCs indicated with the thickness of the red line. Proliferation in the stem cell niche ensures continuous replenishment of the transit-amplifying (TA) compartment. (B) Within the stem cell niche, Lgr5-expressing CBCs are actively dividing and will give rise to both HopX-expressing +4 cells and TA cells. HopX-expressing cells, which are less mitotically active, will give rise to fewer TA cells and occasionally an Lgr5-expressing stem cell. Lrig1 expression in the stem cell niche reduces the amplitude of ErbB activation and is essential for controlling stem cell proliferation.Adult stem cell niches are far more heterogeneous than previously anticipated.¹ The intestinal stem cell niche can be subdivided by the relative position within the crypt. Stem cells located in position +4, just above secretory Paneth cells, express HopX, Bmi1 and Tert. These cells are generally less mitotically active than Lgr5-expressing stem cells located at the bottom of the proliferative crypts intercalated between Paneth cells (Fig. 1A).^2,3 It has been argued that both populations represent the most primitive stem cell; however, recent studies suggest that stem cells can interconvert between the two states (Fig. 1B).³ Fate mapping from cells in position 4 and at the bottom of the crypt supports this.^2,4 The positional cues responsible for cellular sorting into different functional stem cell compartments are poorly characterized. The only known effector of cellular positioning is Wnt (wingless-related MMTV integration site) signaling.⁵ Wnt is highly expressed by Paneth cells along with other mitotic factors, such as ErbB and Notch ligands.⁶ This could functionally account for the differences observed in proliferative potential along the stem cell axis. The discrete expression patterns of Lgr5 and HopX also support the existence of distinct microenvironments that supports cellular identities. A thorough characterization of the factors responsible for stem cell identity will help delineate and define the functional relationship between the distinct stem cell populations.Tissue homeostasis is governed by balanced loss and gain of cells. The stem cell niche supports constant proliferation via pro-mitotic stimuli. In order to control the amplitude of signaling strength, many pathways have developed negative feedback loops. Lrig1 (Leucine-rich repeats and immunoglobulin-like domains 1) is a negative feedback regulator of ErbB-mediated growth factor signaling.⁷ Lrig1 marks stem cells in various epithelial tissues including the intestinal epithelium, where it is expressed within the entire stem cell niche including the +4 and Lgr5-expressing cells (Fig. 1).^8,9 The functional relevance of Lrig1 and negative feedback regulation is clear from the pronounced expansion of the intestinal stem cell compartment observed in the Lrig1-KO mouse model.⁹ This is mediated via increased ErbB signaling and demonstrates the importance of balanced signaling strength within the stem cell niche.⁹ Moreover, an independent study reveals that Lrig1-KO animals have a higher incidence of colorectal cancer, suggesting that unbalanced stem cell proliferation increases tumor susceptibility.¹⁰ Future studies will address whether additional feedback regulators control signaling strength within the intestinal stem cell niche and how homeostasis within the stem cell compartment affects tumor susceptibility.     

Leukocyte transepithelial migration in lung induced by DMSA functionalized magnetic nanoparticles

Magnetic nanoparticles surface-covered with meso-2,3-dimercaptosuccinic acid (MNPs-DMSA) constitute a promising approach for tissue- and cell-targeted delivery of therapeutic drugs in the lung. However, they can also induce a transient transendothelial migration of leukocytes in the organ as a side effect after endovenous administration of MNPs-DMSA. We demonstrated that monocytes/macrophages constitute the main subpopulation of leukocytes involved in this process. Our recent research found that MNPs-DMSA upregulated the mRNA expression of E-, L- and P-selectin and macrophage-1 antigen and increased concentration of tumor necrosis factor α (TNFα) in lung, in a time dependent manner. The critical relevance of the β₂ integrin-dependent pathway in leukocyte transmigration elicited by MNPs-DMSA was demonstrated by use of knockout mice. Our work characterizes mechanisms of the pro-inflammatory effects of MNPs-DMSA in the lung and identifies β₂ integrin-targeted interventions as promising strategies to reduce pulmonary side effects of MNPs-DMSA during biomedical applications. In addition, MNPs-DMSA could be used as modulators of lung immune response.Key words: magnetic nanoparticles, DMSA, nanobiotechnology, transepithelial migration, cell adhesion molecules, integrins, monocytes, lungNanotechnology deals with structures of 100 nm or smaller in at least one dimension and has the potential to create many new materials and devices with a vast range of applications. Materials can be produced that are nanoscale in one dimension (for example, very thin surface coatings), in two dimensions (for example, nanowires and nanotubes) or in all three dimensions (for example, nanoparticles).Magnetic nanoparticles (MNPs) are a class of nanoparticles that can be manipulated using a magnetic field. MNPs are traditionally ferrite-based materials with the general formula MFe₂O₄, where M is a doubly charged metal-ion, such as iron, nickel or cobalt. Magnetic fluids (MFs) are colloidal mixtures composed of MNPs suspended in a carrier fluid, usually an organic or inorganic solvent. There is an increasing interest in developing biocompatible MFs for biomedical applications¹ for instance, for detection of circulating tumor cells,² contrast agents for magnetic resonance imaging³ and in an experimental cancer treatment called magnetic hyperthermia in which the fact that nanoparticles heat when they are placed in an alternative magnetic field is used.⁴ Another potential use includes attaching magnetic nanoparticles to drug/gene for targeting purposes.⁵ In order to be used for medical applications, magnetic nanoparticles are coated with a surfactant to prevent their agglomeration (due to van der Waals and magnetic forces) and allow the association of MNPs surface with different molecules.^6,7In previous studies, we have shown that MNPs surface-coated with meso-2,3-dimercaptosuccinic acid (MNPs-DMSA) (Fig. 1), with average diameter of about 9 nm, presented preferential distribution in the lung tissue, after intravenous administration in mice.^8–10 This target specificity of MNPs-DMSA offers a unique property that may be successfully exploited for the treatment of lung diseases.¹¹ In addition, we reported that the presence of MNPs-DMSA in the lung led to trafficking of leukocytes from blood vessels into pulmonary parenchyma and airspace and that interleukin-1 (IL-1) and interleukin-6 (IL-6) were overexpressed.¹² IL-1 acts as a trigger that activates a cascade of cytokine production and induces the production of a wide range of immunomodulatory cytokines.¹³ IL-6 is among the mediators regulated by IL-1 and is often increased in inflammatory processes in the lung.¹³ These differential expressions were particularly associated with blood vessels and cells of airway ducts suggesting that they could have some role during the recruitment process of inflammatory cells, as observed in histological analyses. In fact, these cytokines are commonly associated with the activation of cells concerning the expression of adhesion surface proteins.¹³ This is in agreement with several studies that described the requirement of IL-1α production in rat airways for full polymorphonuclear cell migration in models for immune-complex deposition or inhalation of cement dust, coal dust or diesel exhaust particles.^14–16Open in a separate windowFigure 1Schematic representation of DMSA-functionalized maghemite nanoparticles.Cell migration plays a key role in a wide variety of biological phenomena. This process is particularly important for leukocyte function and the inflammatory response. A mechanistic understanding of cellular interactions with synthetic surfaces, particularly in the context of inflammatory and healing responses, has been a major goal of biomaterial science.Leukocyte trafficking in the lung involves transendothelial migration, migration in tissue interstitium and transepithelial migration. In addition, leukocyte emigration involves regulatory mechanisms including complement activation, cytokine regulation, chemokine production, activation of adhesion molecules and their respective counter receptors. The process is presumably initiated and modulated by the production of early response cytokines such as IL-1 and tumor necrosis factor (TNF) from lung cells, especially from alveolar macrophages, setting the stage for leukocyte migration through endothelium.¹⁷ On the other hand, ensuing production of interleukin-10 (IL-10) brings into play powerful anti-inflammatory factors that strongly regulate inflammatory responses, functioning as intrinsic regulators of the lung inflammatory response.^18,19Tissue infiltration by circulating leukocytes is a three-step process involving rolling on the endothelium, attachment to the endothelium and transmigration across the endothelial cells lining blood vessel walls (Fig. 2). Leukocyte migration out of the blood is initiated by leukocyte rolling on the luminal side of the endothelium, as mediated by the low-affinity receptors selectins (E-, L- and P-selectin).^20–22 Binding of selectins on leukocytes stimulates “outside-in” signals in these cells, increasing the affinity of the integrin family of receptors (cell surface receptors consisting of an α- and a β-subunit, which are grouped in distinct subfamilies based on β-subunit utilization), which then bind to endothelial cell adhesion molecules such as intercellular adhesion molecule-1 [(ICAM-1)/CD54] and vascular cellular adhesion molecule-1 (VCAM-1). Function-blocking studies have identified the β₁ (CD29) and β₂ (CD18) integrins as the major players involved in leukocyte adhesion and migration.²³ Leukocyte integrin affinity is also rapidly increased by “inside-out” signals from leukocyte chemokine receptors triggered by chemokines displayed on the surface of endothelial cells.²⁴ With an increase in leukocyte integrin receptor affinity, leukocyte rolling is arrested.²⁴Open in a separate windowFigure 2Schematic representation of leukocyte endothelial migration into lung parenchyma.Using immunohistochemistry, we demonstrated that following injection of MF-DMSA, the distribution pattern of E-selectin and members of the β₂ integrin subfamily (macrophage-1 antigen, Mac-1; leukocyte function associated antigen-1, LFA-1) was changed in the lung vessels, but not of β₁ integrin.¹⁰ For L and P selectins no differences were observed between treated and control animals. However, for E-selectin, labeling was found in the endothelium of veins and venules 12 h after MF-DMSA administration, but not in the lung''s vascular compartments of the control and 4 h treatment groups.¹² Concerning integrins, in the control group, leukocytes labeled with Mac-1 and LFA-1 were found only in post-capillary sites. Four hours after MF-DMSA administration, leukocytes expressing these β₂ integrins were also found in capillaries.¹⁰ Our findings expand on other studies showing that the capillary network constitutes an important migration site in the lung.²⁵ Thus, the modulation of Mac-1 and LFA-1 expression in leukocytes located inside capillaries supports the importance of these integrins and capillaries for migratory activity in the lung, in this case after MF-DMSA administration. However, we cannot discard the participation of larger vessels in the migration induced by MNPs-DMSA. In fact, some images from our laboratory have showed that this is also a route used by the leukocytes after injection of these nanoparticles (Fig. 3).Open in a separate windowFigure 3Light microscopy image of leukocytes containing MNPs-DMSA inside a vein. Note that the cells (yellow arrows) are close or attached to the endothelium.It is worth noting that 12 h after MF-DMSA administration, leukocytes labeled with LFA-1 were observed only in post-capillary sites, similar to the control. We speculated that the absence of LFA-1 labeling in capillaries in the period of 12 h after MF-DMSA administration is due to the accentuated decrease of LFA-1 expression levels in the leukocyte over the course of time. In fact, as will be discussed below, we obtained a decrease in the LFA-1 mRNA 12 h after MNPs-DMSA administration. This point of view is in agreement with other studies that demonstrated the distinct contribution of LFA-1 and Mac-1 to transendothelial migration in the lung.²⁶ While both Mac-1 and LFA-1 participate in transendothelial migration at the beginning of the inflammatory process, over time Mac-1 becomes the predominant member of the β₂ integrin subfamily mediating migration of leukocytes.²⁶These results raised several questions related to MNPs-DMSA administration, such as: what is the time profile of leukocyte migration into the airspace? Which is the principal leukocyte subpopulation involved in this process? Is it a fact that the mechanism by which the presence of MNPs-DMSA induces transendothelial migration of leukocytes into the lung is based on their ability to somehow change the expression of cell adhesion molecules on leukocytes and lung vascular endothelial cells? Is β₂ or β₁ integrin, or both, the main receptor involved in MNPs-DMSA leukocyte-induced migration?Recently, we uncovered some of these answers including the main adhesion molecules that are involved in this migration. We first determined that the number of leukocytes in the bronchoalveolar lavage fluid reached its peak 12 h after MNPs-DMSA administration, decreasing to normal values in 48–72 h. Cytologic and FACS analysis demonstrated that the main subpopulation of leukocytes involved in this process was monocyte/macrophage.²⁷It is well known that the reticuloendothelial system, in particular macrophage cells, actively neutralizes and eliminates foreign matter from the body, including nonbiological particles. These and other particulated materials in the lung may lead to lung damage. In fact, transmission electron microscopy analysis clearly demonstrated an uptake of MNPs-DMSA by monocyte/macrophage cells,²⁷ indicating that this may be a mechanism of nanoparticle clearance used by the lung in order to avoid further damage. It is worth noting that an increase in the relative percentage of lymphocytes after MNPs-DMSA administration was also observed. The importance of this finding was not addressed in the paper, but we speculate that it could be important for the control of the inflammatory process initiated by the MNPs-DMSA injection. Failure in control of the inflammatory processes could potentially lead to chronic inflammatory diseases and pulmonary fibrosis. In spite of the fact that we did not determine which was the main source of the production of two different cytokines, one considered pro-inflammatory (TNFα) and the other anti-inflammatory (IL-10), we found an increase in the ratio of IL-10/TNFα cytokine release 12 h after MNPs-DMSA administration. This is clearly a signal that the inflammatory process was being controlled, in agreement with previous reports showing that IL-10 is able to limit the induction of cell adhesion molecules in the lung.²⁸ We presume that lymphocytes are taking part in this process. Further studies are necessary to clarify this point.The nature of the cells present in the pulmonary tissue parenchyma was not determined in this study. However, these cells were not able to cause tissue damage in the lung. We observed no histological or ultrastructural damage in the lung of animals treated with MNPs-DMSA, indicating that the nanoparticle-induced inflammation is not enough to cause chronic disease, such as pulmonary fibrosis.We then determined the effect of MNPs-DMSA on mRNA expression of selectins, integrin β₁ and integrin β₂.²⁷ We found that MNPs-DMSA upregulated the mRNA expression of E-, L- and P-selectin, as well as Mac-1. Further, using knockout mice (deficient in the β₂-subunit common to all β₂ integrins), we observed that, compared to wild-type mice, the recruitment of leukocytes to the airspace following administration of MNPs-DMSA was completely blocked in the former.²⁷ The fact that transmigration of β₂ integrin-deficient monocytes was affected when compared with wild-type monocytes strongly argues in favor of a major contribution by β₂ integrins to monocyte trans-epithelial migration in our system, which is additionally supported by the increase of mRNA of β₂ integrins, as cited above.We should remember, however, that the absence of change in LFA-1 and very late antigen-4 (VLA-4) mRNA does not exclude a role for them in leukocyte migration induced by MNPs-DMSA. Integrins are cell adhesion molecules constitutively expressed on the cell surface and also stored within intracellular vesicles.^29,30 In addition, transendothelial migration of leukocytes depends not only on the number of integrins on the cell surface but also on the change in conformation of these molecules reflecting their activation.³² Therefore, our results did not exclude the possibility that MNPs-DMSA induce the activation of LFA-1 and VLA-4 constitutively located on the surface of leukocytes or the translocation of these integrins from intracellular vesicles to the plasma membrane. On the other hand, the absence of a significant change in the mRNA expression of VCAM-1, which is the major endothelial cell ligand for VLA-4, can be regarded as an indirect indicator that VLA-4 is not involved in this process.The fact that an increase in the mRNA of Mac-1 occurred and there is no change in the mRNA levels of VLA-4 (and LFA-1) corroborates the hypothesis that migration of leukocytes induced by MNPs-DMSA is mainly dependent on β₂ integrins and not β₁ integrins pathway. In addition, we can presume that MAC-1 is the main β₂ integrin molecule involved in the process of leukocyte trafficking.The increased use of nanoparticles in medicine has raised concerns on their ability to gain access to privileged sites in the body. In fact, a study has shown that, in some cases, they can potentially cause damage to tissues located behind cellular barriers. Therefore, it is fundamental to understand the mechanisms underlying interactions between nanoparticles and the body, for their safe and effective use. In the case of MNPs-DMSA, we can use this knowledge for treatment of lung diseases when associated with drugs, as well as for downregulation or upregulation of the local immune system.One important question still unanswered about the use of magnetic nanoparticles in lung disease treatments is what could be expected if more than one dose is necessary in a short period of time. Recent research of Mejias et al.³¹ was close to answer this question. In their study the authors injected repeated doses (nine in total) of magnetic nanoparticles stabilized with DMSA, but unfortunately, they did not analyze the lungs, assuming that the particles would be stocked in the liver, spleen and kidney. For these organs, however, the authors did not refer to any observed damage. We believe that the answer to this question is related with several factors such as physical-chemical features of the nanoparticles (size, hydrodynamic radius, etc.) interval between the injections, amount of iron injected, among others. These features are also important for a second open question: what happens if the organ has a preexistent disease? Further studies are necessary to clarify this point. It is important to minimize, in all cases, the amount of injected iron, increasing, when possible, the amount of drug attached to the nanoparticles. The use of magnetic nanoparticles is already a reality as a contrast agent. It is possible that in the future they also can be used as drug delivery carriers.In resume our work characterizes mechanisms of the pro-inflammatory effects of MNPs-DMSA in the lung and identifies β₂ integrin-targeted interventions as promising strategies to reduce pulmonary side effects of MNPs-DMSA during biomedical applications. In addition, MNPs-DMSA could be used as modulators of lung immune response.     

MEK1/2 and p38-like MAP kinase successively mediate H2O2 signaling in Vicia guard cell

As a second messenger, H₂O₂ generation and signal transduction is subtly controlled and involves various signal elements, among which are the members of MAP kinase family. The increasing evidences indicate that both MEK1/2 and p38-like MAP protein kinase mediate ABA-induced H₂O₂ signaling in plant cells. Here we analyze the mechanisms of similarity and difference between MEK1/2 and p38-like MAP protein kinase in mediating ABA-induced H₂O₂ generation, inhibition of inward K⁺ currents, and stomatal closure. These data suggest that activation of MEK1/2 is prior to p38-like protein kinase in Vicia guard cells.Key words: H₂O₂ signaling, ABA, p38-like MAP kinase, MEK1/2, guard cellAn increasing number of literatures elucidate that reactive oxygen species (ROS), especially H₂O₂, is essential to plant growth and development in response to stresses,^1–4 and involves activation of various signaling events, among which are the MAP kinase cascades.^1–3,5 Typically, activation of MEK1/2 mediates NADPH oxidase-dependent ROS generation in response to stresses,^4,6–8 and the facts that MEK1/2 inhibits the expression and activation of antioxidant enzymes reveal how PD98059, the specific inhibitor of MEK1/2, abolishes abscisic acid (ABA)-induced H₂O₂ generation.^6,8,9 It has been indicated that PD98059 does not to intervene on salicylic acid (SA)-stimulated H₂O₂ signaling regardless of SA mimicking ABA in regulating stomatal closure.^2,6,8,10 Generally, activation of MEK1/2 promotes ABA-induced stomatal closure by elevating H₂O₂ generation in conjunction with inactivating anti-oxidases.Moreover, activation of plant p38-like protein kinase, the putative counterpart of yeast or mammalian p38 MAP kinase, has been reported to participate in various stress responses and ROS signaling. It has been well documented that p38 MAP kinase is involved in stress-triggered ROS signaling in yeast or mammalian cells.^11–13 Similar to those of yeast and mammals, many studies showed the activation of p38-like protein kinase in response to stresses in various plants, including Arabidopsis thaliana,^14–16 Pisum sativum,¹⁷ Medicago sativa¹⁸ and tobacco.¹⁹ The specific p38 kinase inhibitor SB203580 was found to modulate physiological processes in plant tissues or cells, such as wheat root cells,²⁰ tobacco tissue²¹ and suspension-cultured Oryza sativa cells.²² Recently, we investigate how activation of p38-like MAP kinase is involved in ABA-induced H₂O₂ signaling in guard cells. Our results show that SB203580 blocks ABA-induced stomatal closure by inhibiting ABA-induced H₂O₂ generation and decreasing K⁺ influx across the plasma membrane of Vicia guard cells, contrasting greatly with its analog SB202474, which has no effect on these events.^23,24 This suggests that ABA integrate activation of p38-like MAP kinase and H₂O₂ signaling to regulate stomatal behavior. In conjunction with SB203580 mimicking PD98059 not to mediate SA-induced H₂O₂ signaling,^23,24 these results generally reveal that the activation of p38-like MAP kinase and MEK1/2 is similar in guard cells.On the other hand, activation of p38-like MAP kinase^23,24 is not always identical to that of MEK1/2^8,25 in ABA-induced H₂O₂ signaling of Vicia guard cells. For example, H₂O₂^- and ABA-induced stomatal closure was partially reversed by SB203580. The maximum inhibition of both regent-induced stomatal closure were observed at 2 h after treatment with SB203580, under which conditions the stomatal apertures were 89% and 70% of the control values, respectively. By contrast, when PD98059 was applied together with ABA or H₂O₂, the effects of both ABA- and H₂O₂-induced stomatal closure were completely abolished (Fig. 1). These data imply that the two members of MAP kinase family are efficient in H₂O₂-stimulated stomatal closure, but p38-like MAP kinase is less susceptive than MEK1/2 to ABA stimuli.Open in a separate windowFigure 1Effects of SB203580 and PD98059 on ABA- and H₂O₂-induced stomatal closure. The experimental procedure and data analysis are according to the previous publication.^8,23,24It has been reported that ABA or NaCl activate p38 MAP kinase in the chloronema cells of the moss Funaria hygrometrica in 2∼10 min.²⁶ Similar to this, SB203580 improves H₂O₂-inhibited inward K⁺ currents after 4 min and leads it to the control level (100%) during the following 8 min (Fig. 2). However, the activation of p38-like MAP kinase in response to ABA need more time, and only recovered to 75% of the control at 8 min of treatment (Fig. 2). These results suggest that control of H₂O₂ signaling is required for the various protein kinases including p38-like MAP kinase and MEK1/2 in guard cells,^1,2,8,23,24 and the ABA and H₂O₂ pathways diverge further downstream in their actions on the K⁺ channels and, thus, on stomatal control. Other differences in action between ABA and H₂O₂ are known. For example, Köhler et al. (2001) reported that H₂O₂ inhibited the K⁺ outward rectifier in guard cells shows that H₂O₂ does not mimic ABA action on guard cell ion channels as it acts on the K⁺ outward rectifier in a manner entirely contrary to that of ABA.²⁷Open in a separate windowFigure 2Effect of SB203580 on ABA- and H₂O₂-inhibited inward K⁺ currents. The experimental procedure and data analysis are according to the previous publication.²⁴ SB203580 directs ABA- and H₂O₂-inactivated inward K⁺ currents across plasma membrane of Vicia guard cells. Here the inward K⁺ currents value is stimulated by −190 mV voltage.Based on the similarity and difference between PD98059 and SB203580 in interceding ABA and H₂O₂ signaling, we speculate the possible mechanism is that the member of MAP kinase family specially regulate signal event in ABA-triggered ROS signaling network,^1–4 and the signaling model as follows (Fig. 3).Open in a separate windowFigure 3Schematic illustration of MAP kinase-mediated H₂O₂ signaling of guard cells. The arrows indicate activation. The line indicates enhancement and the bar denotes inhibition.     

Quantitative real-time imaging of molecular dynamics during cancer cell invasion and metastasis in vivo

Despite our advanced understanding of primary cancer development and progression, metastasis and the systemic spread of the disease to secondary sites remains the leading cause of cancer-associated death. The metastatic process is therefore a major potential therapeutic target area for cancer researchers and elucidating the key steps that are susceptible to therapeutic intervention will be critical to improve our treatment strategies. Recent advances in intravital imaging are rapidly improving our insight into this process and are helping in the design of stage-specific drug regimes for the treatment of metastatic cancer. Here we discuss current developments in intravital imaging and our recent use of photobleaching and photoactivation in the analysis of dynamic biomarkers in living animals to assess the efficacy of therapeutic intervention on early stages of tumor cell metastasis.Key words: in vivo imaging, photobleaching, photoactivation, biomarkersMetastasis is a complex process consisting of interactions between cancer cells and their surrounding extra-cellular matrix and stroma. To give rise to a secondary tumor, a primary tumor cell undergoes alterations to its cell-cell and cell-ECM contacts, allowing it to breach the basement membrane and intravasate into the vasculature or the lymphatic system. A tumor cell must survive in the circulation before extravasating at a secondary site and initiating new tumor growth and the development of its own blood supply. Imaging this process in live animals under native physiological conditions is inherently difficult due to poor sample stability, tissue penetration and autofluorescence of the tissue. However, new advances in fluorescent imaging, including the continued development of green fluorescent protein (GFP) and its variants, have facilitated the observation of this process and shed light on some key mechanisms that determine how and why cells metastasise. The use of fluorescent probes for in vivo imaging can be divided into two types (1) ‘passive’ markers or reporters used for direct visualization and tracking of cell movement in relation to extracellular structures and (2) more complex, ‘active’ reporters or biosensors for monitoring detailed processes such as biochemical activity or protein-protein interactions during metastasis.^1,2 In some cases there can be overlap between both types of imaging which will be addressed here.The majority of early intravital imaging studies focused on the stages of metastasis that occur after dissemination from the primary tumor and predominantly used a ‘passive’ reporter approach to assess tumor cell behavior. Models of circulating tumor cells have allowed for analysis at the single cell level of tumor cell velocity, persistence, shape change and interactions with the ECM and stroma in secondary tissue.^3–5 The use of fluorescently-labelled cells has also revealed some limiting factors that cause the arrest of cancer cells in target tissue such as trapping in small capillary networks due to tumor cell size or adhesion to surrounding vessel walls.^6,7 Furthermore, experimental models of metastasis such as intra-splenic, intra-cardial and tail vein injections in combination with fluorescently-tagged tumor cells has provided information on the colonisation, extravasation and dormancy of tumor cells in secondary sites (Fig. 1 and refs. ^{5, 8} and ⁹). Collectively, along with the rapid increase in tissue specific expression of GFP in mouse cancer models,¹⁰ a wealth of information on different steps of the metastatic process has begun to emerge.Open in a separate windowFigure 1(A) Whole body optical imaging of mCherry-expressing SW 620 colon cancer cell metastases after approximately six weeks post intra-splenic injection. Images were obtained using the Olympus OV100 whole body imaging system with an Olympus MT10, 150 w, Xenon light source, using a low magnification objective (macro lens) with a magnification of 0.14× and numerical aperture of 0.04. (B) mCherry expressing SW 620 colon cancer cells colonizing the liver 30 mins after intra-splenic injection. 1 × 10⁶ cells were injected into the spleen of an anesthetised CD-1 nude mouse and the incision sealed using ‘Clay Adams’ vetinary clips (VetTec). The mouse was placed on a heat pad for 30 mins then sacrificed. An incision was made in the abdomen to expose the liver and images of fluorescent cells within the liver were obtained using a 0.8× (0.22 NA) objective lens with variable zoom on the Olympus OV100.The departure of individual cells away from solid primary tumors into the blood stream has been a more difficult process to study using intravital imaging. It is a rare, sporadic event, requiring long acquisition and the inherent density and complex nature of the tumor tissue poses problems for imaging. Overcoming autofluorescence and light scattering has recently been improved due to advances in fluorophores^1,11 and the combined use of long-term multiphoton microscopy¹² has allowed greater resolution and tissue penetration than before. Multiphoton imaging can also provide additional detail regarding the interaction between cells and the surrounding extracellular environment using second harmonic signal generation (SHG) from collagen, elastin and other matrix proteins found in connective tissue.^13,14 In this regard, imaging the interaction of cancer cells with extracellular matrix has revealed distinct modes of cell locomotion adopted by cancer cells in vivo, such as ameboid or mesenchymal invasion, that depend upon the topography or density of the surrounding matrix.^3,13,15 A greater understanding of the initial cell movement and interaction with the extracellular environment will enhance our ability to pin-point cell-ECM targets that may be of clinical relevance in the future.Concurrent with the use of GFP as a ‘passive’ marker, a number of techniques have been developed that facilitate the visualization and localisation of GFP-tagged fusion proteins to quantify changes in protein expression, mobility and sub-cellular interactions during various processes in vitro. These include photobleaching (PB), photoactivation (PA), fluorescence resonance energy transfer (FRET) and fluorescent life time imaging microscopy (FLIM).^2,16,17 The adaptation of these techniques for in vivo imaging to examine the activity of key molecules will provide new ‘active’ markers or reporters that can be correlated with biological processes important in disease progression such as migration, proliferation and cell death. Other fluorescent probes such as MMPsense or Apotrace that measure ‘active’ processes such as metalloproteinase activity or apoptosis have also recently been used in animals.^18,19 In this way we can get closer to understanding how subcellular components or signal transduction pathways interact in real-time. The improved spatial and temporal detail will facilitate the ‘when and where’ we should target metastatic cancer cells for therapy.¹²In our recent paper we have adapted two techniques, photobleaching and photoactivation, for in vivo imaging and used them to assess the potential of E-cadherin as a molecular biosensor for cell migration in live tumors.²⁰ E-cadherin-based cell-cell contacts are prominent sites of remodelling during early stages of epithelial to mesenchymal transition (EMT). The disruption or deregulation of E-cadherin-based adhesions leads to the collapse of normal epithelial architecture that precedes the initial intravasation of cells from tumors.^21–23 In vitro photobleaching analysis of E-cadherin can be used as an ‘active’ molecular read-out of cell migration, as cells within a stationary colony show significantly reduced E-cadherin mobility compared to collectively migrating cells.²⁰ Moreover, as demonstrated in Figure 2 (reviewed in ref. ²⁰), E-cadherin mobility can also be spatially regulated within a population of tumor cells, as cells at the rear of a wound show impaired E-cadherin mobilisation compared to cells at the leading edge of the wound. This suggests a gradient of E-cadherin mobilisation within the local environment of a tumor may exist and could potentially be used in the future to map areas of weakened cell-cell adhesion from which cells are more likely to migrate. In vivo analysis of E-cadherin dynamics showed that changes in the mobility of E-cadherin can also be used as an ‘active’ marker of cell behavior in live animals, and may be useful in predicting cell mobilisation from primary tumors.²⁰Open in a separate windowFigure 2FRAP of GFP-E-cadherin at the rear or front of a wound heal assay. (A and B) Schematic and representative images of a wound heal assay depicting the area of cells selected for E-cadherin-based cell-cell junction FRAP analysis (red broken line). (C and D) Representative images of FRAP experiments performed at the rear or front of a wound heal assay respectively. White solid arrows represent area of photobleaching at the rear and white broken arrows represent area of photobleaching at the front of the wound. Red arrows indicate dynamics of cells at the front of the wound. Cells were classed to be at the front of the wound within the first three cells from the wound border (reviewed in ref. ²⁰).We also demonstrated the subcellular tracking of plasma membrane dynamics in vivo using the membrane-targeting sequence of H-Ras fused to photoactivatable-GFP.^24,25 Importantly, both the dynamics of cell-cell junctions, as visualised using E-cadherin:GFP, and the dynamics of the plasma membrane, which also plays a fundamental role in cell invasion and metastasis, are significantly different in vivo than in vitro.²⁰ Critically, this raises the possibility that many signalling axes and networks may function differently in vivo and therefore care must be taken when correlating in vitro information to the live setting. Lastly, we demonstrated the benefits of in vivo imaging in the assessment of molecular-based targeted therapeutics by using the Src inhibitor dasatinib, which impaired E-cadherin cell mobility in vivo but not in vitro.^20,26In the context of previous intravital imaging studies, our work suggests that we are at the beginning of a new stage of intravital imaging in which ‘active’ probes can help predict the efficacy of novel therapeutic treatments and also provide a context dependent read-out of oncogene-induced biological behavior in live animals. Importantly, not all molecules are adaptable for this type of in vivo imaging. Careful selection of candidate molecular markers that demonstrate clear changes attributable to a biological function, for example, subcellular relocalization or compartmentalisation, will be ideally suited for this type of intravital examination in the future.Here we have adopted two key fluorescent imaging techniques typically used in vitro and combined them with a fundamental biological question in vivo. The adaptation of other techniques for in vivo imaging such as FRET or FLIM-FRET probes will provide a detailed pixel by pixel map of the activity and behavior of key signalling proteins in live animals.^2,27 The use of these ‘active’ probes in vivo may hold further surprises concerning differences in molecular behavior in live animals compared to the traditional ‘snap-shot’ approach in vitro. Finally, one of the major challenges of in vivo imaging during drug discovery is the need for repeated imaging of the same animal in the presence or absence of drugs. The continued development of optical windows and observation chambers for non-invasive real-time imaging will facilitate this and allow for the assessment of drug response at the single cell level.²⁸ This, when combined with the subcellular optical techniques described here, will prove very useful in the future for in vivo imaging when evaluating the aetiology of the disease or during the drug discovery process.     

Collective invasion of carcinoma cells: When the fibroblasts take the lead

Epithelial to mesenchymal transitions (EMT) have been suggested to be crucial during epithelial cancer cell invasion. However, in a three-dimensional “organotypic” invasion assay squamous cell carcinoma (SCC) cells that retain epithelial characteristics “hitch a ride” with carcinoma associated fibroblasts (CAFs) in order to collectively invade. Thus epithelial cancer cells can utilise the mesenchymal characteristics of CAFs without the need to undergo EMT themselves. This work provides new insight in cancer cell invasion and shows a new role for CAFs as a target for an anti-invasive therapy.Key words: collective invasion, carcinoma associated fibroblast, extracellular matrix, matrix metalloproteinases, RhoCancer cell invasion and metastasis are the main causes of mortality in cancer patients. Understanding how cancer cells move and invade within the surrounding tissue is therefore a key issue. Stromal fibroblasts within a tumor play a crucial role in cancer cell proliferation, survival, angiogenesis as well as invasion (reviewed in ref. ¹). In many cases stromal CAFs are able to produce a wide range of growth factors and cytokines that modulate tumor growth and invasion.^2,3 Their influence in cancer cell invasion and metastasis can also be mediated through the production of MMP''s that promote extra-cellular matrix degradation.⁴It has recently been shown that CAFs can play an unexpected role in SCC invasion.⁵ In a 3D ‘organotypic’ model of invasion that recreates the epidermal/dermal environment CAFs promote the collective invasion of SCC cells.⁶ 3D time-lapse confocal microscopy imaging showed that CAFs were always the leading cell of the invading cohort with the SCC cells following behind. These cohorts closely resembled invading clusters of SCC cells observed in human cancer samples.⁷ CAFs promoted SCC cells collective invasion by remodelling the matrix and making a path that SCC cells can use to invade. This process is clearly shown in Figure 1: a CAF (in red) leads the invasion of a collective chain of SCC cells (green) and makes a path in the surrounding matrix, visualized in grey using confocal reflectance microscopy. Two key experiments helped to understand the role of fibroblasts in this system. Firstly, the separation of the two cell populations by a thin layer of gel without fibroblasts completely abolished SCC invasion and so ruled out the possibility of long distance chemoattractant molecules inducing SCC invasion. Secondly, SCC cells were able to invade into a gel which had previously been remodelled by CAFs that had subsequently been removed. Together these experiments showed that tracks made by the fibroblasts are essential and sufficient to promote collective carcinoma cells invasion. Heterotypic cell contact between both populations was not required, as SCC cells can invade using tracks made by the CAFs even if the CAFs have been removed.Open in a separate windowFigure 1Collective invasion of carcinoma cells led by fibroblast. Confocal time-lapse imaging of carcinoma associated fibroblast (red) leading the way of an invading chain of SCC cells (green) and making path into the surrounding matrix (grey). Panel is 80 x 80 mm and spans 300 minutes, scale are 20 um.Interestingly, inhibition of Rho/ROCK signalling to the actomyosin cytoskeleton or MMPs using small molecule inhibitors blocked SCC invasion even when only CAFs where targeted. Blocking these pathways in carcinoma cells had little or no effect on their invasion. Moreover, inhibition of Rho function specifically in CAFs did not block their invasion into matrices but prevented SCC cells from following. These experiments showed the role of Rho/ROCK and MMPs molecular pathways in track generation by the CAFs and that targeting these pathways in CAFs, but not SCC cells, is critical for preventing cancer invasion. Strikingly, blockade of protease function after CAFs had remodelled the ECM had little effect on the ability of SCC cells to invade. This could explain the relative poor results obtained using MMPs inhibitors as anti-invasive therapies.⁸ Rho/ROCK function was dispensable in SCC cells; however, depletion of the small GTPase Cdc42 and its effector MRCK disrupted the acto-myosin cortex of carcinoma cells and blocked their capacity to invade in response to CAFs.In order to invade and metastasise, carcinoma cells can switch from an epithelial state to a more mesenchymal phenotype.⁹ This process, called EMT, allows epithelial cancer cells to adapt their behaviour and confers the capacity to remodel the ECM on the cancer cells.¹⁰ However, in patient tissue samples, it has been observed that carcinoma cells can invade without undergoing an EMT, these cancer cells do not upregulate mesenchymal markers and retain cell to cell contact during their invasion.¹¹ This work explains how carcinoma cells that have not undergone EMT could invade a 3D matrix. These cells use the mesenchymal characteristics of the stromal fibroblasts to remodel the ECM and consequently follow behind invading fibroblasts. In tumours of mesenchymal origin CAFs are not required for invasion; work from Friedl and colleagues, clearly shows that HT1080 fibrosarcoma cells could lead collectively invading chains of cancer cells The authors showed how the leading cell of the collective chain remodels collagen fibres into tracks as it invades through the action of MT1-MMP (MMP14).¹²In normal conditions, epithelial cells and dermal fibroblasts are in complete homeostasis and separated by a basement membrane (Fig. 2A). In addition, normal dermal fibroblasts are unable to promote SCC invasion. Understanding how CAFs are activated will be an important step forward. A desmoplastic response is observed in many tumours indicating a change in behaviour of fibroblasts.¹³ During wound healing or fibrosis, fibroblasts are in an active state that has been suggested to be similar to cancer activation.¹⁴ TGFβ has been shown to be a key player in fibroblasts activation and could support cancer progression.¹⁵ However, TGFβ was not responsible for SCC cells invasion since a TGFβ inhibitor had no effect in carcinoma cells collective invasion induced by the CAFs in the 3D invasion assay (Cedric Gaggioli and Steven Hooper, unpublished data). Interestingly, a probe that binds only to the active form of the small GTPase Rho showed that the activity of this protein was increased in CAFs compared to normal fibroblasts in tissue samples. Elevated expression of α5 integrin was also present in these cells and this has been implicated in Rho activation in a number of systems.^16–18 Consistent with this observation, depletion of integrin a5 in CAFs reduced their ability to promote the invasion of SCC cells. Alternatively, CAFs could also be derived from endothelial cells through a process called endothelial to mesenchymal transition¹⁹ (EndMT), or from cancer cells through EMT.²⁰ These processes could be responsible for CAFs generation in the tumor stroma resulting in matrix remodelling and tracks generation in order for the carcinoma cells to collectively invade the surrounding tissue and metastasize (Fig. 2B).Open in a separate windowFigure 2Model of carcinoma cells collective invasion. (A) Schematic representation of a normal epithelium. Epithelial cells (light blue) and normal fibroblasts (pink) are separated by a basal membrane and are in a perfect homeostasis. Cross talk between both cell types occurs through adhesion and chemokine secretion. (B) Schematic representation of carcinoma cells collective invasion. CAFs (red) take the lead of a collective invading chain of SCC cells (brown). Invasion of CAFs is MMPs dependent but Rho/ROCK independent. However, track generation by CAFs is Rho/ROCK/MLC dependent. SCC cells require the small GTPase Cdc42 and its effector MRCK in order to collectively invade trough those tracks (black).This study opens a new field of investigation for collective cancer cell invasion. This work highlights carcinoma associated fibroblasts as new therapeutic targets which will be a new direction in cancer cell invasion and metastasis therapy.     

Correlation between the levels of circulating adhesion molecules and atherosclerosis in type-2 diabetic normotensive patients: Circulating adhesion molecules and atherosclerosis

Endothelial dysfunction is a common feature in type-2 diabetic patients and is associated with inflammation, increased levels of circulating soluble adhesion molecules and atherosclerosis. The aim of this study was to evaluate the relationship between the levels of circulating soluble adhesion molecules and the degree of atherosclerosis in normotensive type-2 diabetic patients.Results: We found significant correlations between ICAM-1 (r = 0.69, p < 0.001 95% IC 0.65 to 0.82) and VCAM-1 (r = 0.4, p < 0.03, 95% IC 0.65 to 0.82) levels and maximal carotid artery intimal-medial thickness, whereas no correlation was observed with E-selectin.Methods: We studied 30 normotensive type-2 diabetic patients in whom VCAM-1, ICAM-1 and E-selectin were measured by ELISA. Additionally, the intimal-medial thickness of both the common and internal carotid arteries was measured (B-mode ultrasound). The levels of circulating adhesion molecules and maximal carotid artery intimal-medial thicknesses were correlated using the Spearman correlation coefficient test. Statistical analysis was performed with ANOVA.Conclusion: Our results suggest that ICAM-1 and VCAM-1 are markers associated, and correlated with the degree of atherosclerosis in normotensive type-2 diabetic patients.Key words: atherosclerosis, soluble adhesion molecules, normotensive type-2 diabetic patientsType-2 diabetic patients suffer from some form of endothelial dysfunction. Endothelial dysfunction in these patients stimulates inflammation and increases levels of circulating soluble adhesion molecules.¹ Both endothelial dysfunction and inflammation contribute to atherosclerosis via several mechanisms.²Leukocytes are unable to adhere to normally functioning arterial endothelium; however, in the setting of endothelial dysfunction, the bioavailability of nitric oxide is reduced, resulting in the activation of nuclear factor κB (NFκB). NFκB increases proinflammatory gene expression, including the expression of leukocyte adhesion molecules,³ which are expressed on the arterial endothelium.⁴ Also, NFκB increases systemic concentrations of soluble forms of adhesion molecules (SAM), perhaps as a result of a proteolytic processing on the endothelial cell surface.^4,5 Circulating levels of SAM are thought to reflect increased endothelial cell surface expression⁵ and high serum levels of SAM are considered markers of endothelial dysfunction.¹ Endothelial dysfunction seems to be the trigger in atherogenesis and diabetes-associated vascular disease and explains, at least in part, the enhanced progression of CVD (cardiovascular disease) in type 2 diabetes.Several inflammatory markers, such as C reactive protein and interleukin 6, have been implicated in diabetic macrovascular complications.^2,4 Early atherosclerosis has an inflammatory component characterised by leucocytic infiltration of the vascular endothelial wall,⁶ SAMs may be implicated in the development of the atherosclerotic plaque by facilitating the attachment and migration of leukocytes into the arterial wall, which is a critical early step in the initiation of atherosclerosis.^3,5 In fact, genetically modified mice that lack expression of adhesion molecules do not develop atherosclerosis and the administration to mice of antibodies directed against SAM decreases intimal hyperplasia.⁶ Adhesion molecules have been observed consistently within the milieu of the atherosclerotic plaque,^2,7 but it is not clear whether circulating levels of SAM are associated with atherosclerosis, specifically in type-2 diabetic patients, in whom circulating levels of SAM are increased.^8,9High-resolution B-mode ultrasonography provides a noninvasive method of quantifying subclinical arterial wall thickening and atherosclerotic progression. Ultrasound is preferable to arteriography because it is noninvasive, carries no risk for the examined subject, and can detect atherosclerosis as an increase in arterial wall thickness before a reduction in lumen diameter occurs. Increases in the thickness of the intimal-medial layer are measured by ultrasonography and have been directly associated with an increased risk of myocardial infarction and stroke.¹⁰The aim of this study was to evaluate whether relationships between circulating soluble adhesion molecules levels and the degree of atherosclerosis (defined as the maximal intimal-medial thickness) exist in normotensive type-2 diabetic patients.