CTGF-mediated autophagy-senescence transition in tumor stroma promotes anabolic tumor growth and metastasis

Comment on: Capparelli C, et al. Cell Cycle 2012; 11:2272-84 and Capparelli C, et al. Cell Cycle 2012; 11:2285-302.Otto Warburg first observed that cancer cells preferentially undergo glycolysis instead of the mitochondrial TCA cycle even under oxygen-rich conditions. This form of energy metabolism in cancer cells is called “aerobic glycolysis” or the “Warburg effect.”¹ Lisanti and colleagues have previously proposed an alternative model called the “the reverse Warburg effect,” in which aerobic glycolysis predominantly occurs in stromal fibroblasts.² During this process, cancer cells secrete oxidative stress factors, such as hydrogen peroxide, into the tumor microenvironment, which induces autophagy. This leads to degradation of mitochondria (mitophagy) and elevated glycolysis in cancer-associated fibroblasts.³ Aerobic glycolysis results in the elevated production of pyruvate, ketone bodies and L-lactate, which can be utilized by cancer cells for anabolic growth and metastasis. At the molecular level, stromal fibroblasts lose expression of caveolin-1 and activate HIF-1a (Fig. 1), TGFβ and NFκB signaling.⁴ Stromal caveolin-1 expression predicts clinical outcome in breast cancer patients.⁵Open in a separate windowFigure 1. CTGF-mediated autophagy-senescence transition in tumor stroma promotes anabolic tumor growth and metastasis. Cancer cells secrete oxidative stress factors (H₂O₂) that induce autophagy in cancer-associated fibroblasts. Additionally, caveolin-1 (cav-1) loss leads to activation of connective tissue growth factor (CTGF) and HIF-1α that mediate autophagy and senescence in these stromal cells. This is called the autophagy-senescence transition (AST). AST leads to mitophagy and elevated glycolysis in cancer-associated fibroblasts. Aerobic glycolysis results in the elevated production of several nutrients (pyruvate, ketone bodies and L-lactate), which can be utilized by cancer cells for tumor growth and metastasis.In the June 15, 2012 issue of Cell Cycle, two studies by Capparelli et al. further validate the “autophagic tumor stroma model of cancer” described above, as well as identify novel mechanisms involved in this process.^6,7 Autophagy and senescence are induced by the same stimuli and are known to occur simultaneously in cells. In the first study, the authors hypothesize that the onset of senescence in the tumor stroma in response to autophagy/mitophagy contributes to mitochondrial dysfunction and aerobic glycolysis. In order to genetically validate this process of autophagy-senescence transition (AST) (Fig. 1), Capparelli et al. overexpressed several autophagy-promoting factors (BNIP3, cathepsin B, Beclin-1 and ATG16L1) in hTERT fibroblasts to constitutively induce autophagy. Autophagic fibroblasts lost caveolin-1 expression and displayed enhanced tumor growth and metastasis when co-injected with breast cancer cells in mice, without an increase in angiogenesis. In contrast, constitutive activation of autophagy in breast cancer cells inhibited in vivo tumor growth. Autophagic fibroblasts also showed mitochondrial dysfunction, increased production of nutrients (L-lactate and ketone bodies) and features of senescence (β-galactosidase activity and p21 activation). AST was also demonstrated in human breast cancer patient samples.⁷ In the second study, using a similar experimental approach, the authors evaluated the role of the TGFβ target gene, connective tissue growth factor (CTGF), in the induction of AST and aerobic glycolysis in cancer-associated fibroblasts. CTGF would be activated in the tumor stroma upon loss of caveolin-1. CTGF overexpression in fibroblasts induced autophagy/mitophagy, glycolysis and L-lactate production in a HIF-1α-dependent manner along with features of senescence and oxidative stress. CTGF overexpression in fibroblasts also promoted tumor growth when co-injected with breast cancer cells in mice (Fig. 1), independent of angiogenesis. As expected, CTGF overexpression in breast cancer cells inhibited tumor growth. CTGF is known to be involved in extracellular matrix synthesis; however, the effects of CTGF overexpression in fibroblasts and tumor cells were found to be independent of this function.⁶Overall, the authors have identified a novel mechanism by which CTGF promotes AST and aerobic glycolysis in cancer-associated fibroblasts. In turn, the stromal cells stimulate anabolic tumor growth and metastasis. The authors also genetically validate the two-compartment model of cancer metabolism, whereby autophagy genes and CTGF have differential effects in stromal cells and tumor cells. The current studies have several implications for cancer therapy. The finding that HIF-1 activation is necessary for the induction of autophagy and senescence downstream of caveolin-1 loss and CTGF activation in stromal fibroblasts is intriguing. Activation of HIF-1 in the hypoxic tumor microenvironment is known to promote tumor cell growth, survival and therapeutic resistance.⁸ Therefore, targeting HIF-1 has the potential to block tumor progression through dual inhibitory effects on hypoxic cancer cell growth and survival as well as the induction of autophagy in stromal fibroblasts. CTGF and AST in the tumor stroma could serve as biomarkers for predicting clinical outcome, therapy response and metastasis. The two-compartment model of tumor metabolism raises further questions regarding the use of antioxidants and autophagy inhibitors/inducers for cancer therapy. The use of these agents in the clinic should be carefully evaluated considering their differential effects on stromal cells and cancer cells.     

“Double hit” makes the difference

Comment on: Menendez JA, et al. Cell Cycle 2012; 11:2782–92.Metformin (N’, N’-dimethylbiguanide) is an anti-diabetic drug prescribed to more than 100 million patients in the world. In addition to its efficacy for the treatment of diabetes, several recent studies have shown that it has anti-tumoral properties.¹ We and others have shown that metformin targets cancer cell metabolism by inhibiting mitochondrial complex 1 activity.^2,3 This energetic stress leads to a decrease of intracellular ATP concentration, and cancer cells will increase their rate of glycolysis.² This compensatory response is not sufficient to restore ATP levels, but is adequate to maintain viable cells in most of the cancer cells. Indeed, metformin blocks cell growth but can also induce apoptosis in some cancer cell models.⁴ The increase of glycolysis induced by metformin is somehow inconsistent with the observed inhibition of proliferation, since cancer cells use preferentially glycolysis to grow faster. This switch to glycolysis, also known as the “Warburg effect,” is linked to oncogenic transformation⁵ and is accompanied by the hyperactivation of the mTOR pathway. In cancer cells, the increase of glycolysis induced by metformin is associated with a strong inhibition of the mTOR pathway via the AMPK. This new metabolic order established by metformin may explain the paradoxical effect of metformin. In view of the above scenario, Menendez et al. decided to test the synthetic lethality of metformin and combined metformin treatment with glucose starvation. They showed that the treatment of breast cancer cells with metformin alone does not induce apoptosis but arrests cells in G₀/G₁. Glucose starvation by itself induces few apoptosis, but the combination of metformin with the absence of glucose induces massive apoptosis. This is not altogether surprising, since the dual action of metformin and glucose starvation block the two main ways of production of ATP (i.e., mitochondrial respiration and glycolysis) (Fig. 1). This is an interesting observation, which could be valuable for future anticancer therapy; however, glucose starvation is not therapeutically feasible. Thus, the use 2-deoxyglucose (2-DG), an inhibitor of glycolysis, could be useful. We and others found that the combination of 2-DG and metformin inhibits prostate cancer cell proliferation and breast tumor growth in xenograft models.^2,6 Although it induces a slight apoptotic response in vitro, 2-DG alone is not efficient in vivo to alter tumor growth⁶ but improves the curative action of radiotherapy;⁷ similarly, it reinforces metformin action. Another interesting issue raised by Menendez et al. is the use of such dual therapy to target cancer stem cells. Metformin has been shown to selectively kill cancer stem cells and the chemotherapy-resistant subpopulation of cancer stem cells.^8,9 Cancer stem cells greatly depend on aerobic glycolysis to sustain their stemness and immortality. The synthetic lethality induced by metformin and glucose starvation may help to improve chemotherapy action and avoid cancer relapse. In conclusion, targeting cancer cell metabolism with a “dual hit therapy” opens new avenues for the future treatment of cancer.Open in a separate windowFigure 1. The combination of metformin and glucose starvation induces a strong energetic stress. Metformin inhibits the mitochondrial complex 1 and glucose starvation, or 2-DG inhibits ATP production from glycolysis. The combination of the two energetic stresses induces a massive energetic stress and leads to a strong apoptotic response.     

Plant defensins: Defense,development and application

Plant defensins are small, highly stable, cysteine-rich peptides that constitute a part of the innate immune system primarily directed against fungal pathogens. Biological activities reported for plant defensins include antifungal activity, antibacterial activity, proteinase inhibitory activity and insect amylase inhibitory activity. Plant defensins have been shown to inhibit infectious diseases of humans and to induce apoptosis in a human pathogen. Transgenic plants overexpressing defensins are strongly resistant to fungal pathogens. Based on recent studies, some plant defensins are not merely toxic to microbes but also have roles in regulating plant growth and development.Key words: defensin, antifungal, antimicrobial peptide, development, innate immunityDefensins are diverse members of a large family of cationic host defence peptides (HDP), widely distributed throughout the plant and animal kingdoms.^1–3 Defensins and defensin-like peptides are functionally diverse, disrupting microbial membranes and acting as ligands for cellular recognition and signaling.⁴ In the early 1990s, the first members of the family of plant defensins were isolated from wheat and barley grains.^5,6 Those proteins were originally called γ-thionins because their size (∼5 kDa, 45 to 54 amino acids) and cysteine content (typically 4, 6 or 8 cysteine residues) were found to be similar to the thionins.⁷ Subsequent “γ-thionins” homologous proteins were indentified and cDNAs were cloned from various monocot or dicot seeds.⁸ Terras and his colleagues⁹ isolated two antifungal peptides, Rs-AFP1 and Rs-AFP2, noticed that the plant peptides'' structural and functional properties resemble those of insect and mammalian defensins, and therefore termed the family of peptides “plant defensins” in 1995. Sequences of more than 80 different plant defensin genes from different plant species were analyzed.¹⁰ A query of the UniProt database (www.uniprot.org/) currently reveals publications of 371 plant defensins available for review. The Arabidopsis genome alone contains more than 300 defensin-like (DEFL) peptides, 78% of which have a cysteine-stabilized α-helix β-sheet (CSαβ) motif common to plant and invertebrate defensins.¹¹ In addition, over 1,000 DEFL genes have been identified from plant EST projects.¹²Unlike the insect and mammalian defensins, which are mainly active against bacteria,^2,3,10,13 plant defensins, with a few exceptions, do not have antibacterial activity.¹⁴ Most plant defensins are involved in defense against a broad range of fungi.^2,3,10,15 They are not only active against phytopathogenic fungi (such as Fusarium culmorum and Botrytis cinerea), but also against baker''s yeast and human pathogenic fungi (such as Candida albicans).² Plant defensins have also been shown to inhibit the growth of roots and root hairs in Arabidopsis thaliana¹⁶ and alter growth of various tomato organs which can assume multiple functions related to defense and development.⁴     

Matrix metalloproteinase (MMP) and TGFβ1-stimulated cell migration in skin and cornea wound healing

Cell migration during wound healing is a complex process that involves the expression of a number of growth factors and cytokines. One of these factors, transforming growth factor-beta (TGFβ) controls many aspects of normal and pathological cell behavior. It induces migration of keratinocytes in wounded skin and of epithelial cells in damaged cornea. Furthermore, this TGFβ-induced cell migration is correlated with the production of components of the extracellular matrix (ECM) proteins and expression of integrins and matrix metalloproteinases (MMPs). MMP digests ECMs and integrins during cell migration, but the mechanisms regulating their expression and the consequences of their induction remain unclear. It has been suggested that MMP-14 activates cellular signaling processes involved in the expression of MMPs and other molecules associated with cell migration. Because of the manifold effects of MMP-14, it is important to understand the roles of MMP-14 not only the cleavage of ECM but also in the activation of signaling pathways.Key words: wound healing, migration, matrix metalloproteinase, transforming growth factor, skin, corneaWound healing is a well-ordered but complex process involving many cellular activities including inflammation, growth factor or cytokine secretion, cell migration and proliferation. Migration of skin keratinocytes and corneal epithelial cells requires the coordinated expression of various growth factors such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), transforming growth factor (TGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), epidermal growth factor (EGF), small GTPases, and macrophage stimulating protein (reviewed in refs. ¹ and ²). The epithelial cells in turn regulate the expression of matrix metalloproteinases (MMPs), extracellular matrix (ECM) proteins and integrins during cell migration.^1,3,4 TGF-β is a well-known cytokine involved in processes such as cell growth inhibition, embryogenesis, morphogenesis, tumorigenesis, differentiation, wound healing, senescence and apoptosis (reviewed in refs. ⁵ and ⁶). It is also one of the most important cytokines responsible for promoting the migration of skin keratinocytes and corneal epithelial cells.^3,6,7TGFβ has two quite different effects on skin keratinocytes: it suppresses their multiplication and promotes their migration. The TGFβ-induced cell growth inhibition is usually mediated by Smad signaling, which upregulates expression of the cell cycle inhibitor p21^WAF1/Cip1 or p12^CDK2-AP1 in HaCaT skin keratinocyte cells and human primary foreskin keratinocytes.^8,9 Keratinocyte migration in wounded skin is associated with strong expression of TGFβ and MMPs,¹ and TGFβ stimulates the migration of manually scratched wounded HaCaT cells.¹⁰ TGFβ also induces cell migration and inhibits proliferation of injured corneal epithelial cells, whereas it stimulates proliferation of normal corneal epithelial cells via effects on the MAPK family and Smad signaling.^2,7 Indeed, skin keratinocytes and corneal epithelial cells display the same two physiological responses to TGFβ during wound healing; cell migration and growth inhibition. However as mentioned above, TGFβ has a different effect on normal cells. For example, it induces the epithelial to mesenchymal transition (EMT) of normal mammary cells and lens epithelial cells.^11,12 It also promotes the differentiation of corneal epithelial cells, and induces the fibrosis of various tissues.^2,6The MMPs are a family of structurally related zinc-dependent endopeptidases that are secreted into the extracellular environment.¹³ Members of the MMP family have been classified into gelatinases, stromelysins, collagenases and membrane type-MMPs (MT-MMPs) depending on their substrate specificity and structural properties. Like TGFβ, MMPs influence normal physiological processes including wound healing, tissue remodeling, angiogenesis and embryonic development, as well as pathological conditions such as rheumatoid arthritis, atherosclerosis and tumor invasion.^13,14The expression patterns of MMPs during skin and cornea wound healing are well studied. In rats, MMP-2, -3, -9, -11, -13 and -14 are expressed,¹⁵ and in mice, MMP-1, -2, -3, -9, -10 and -14 are expressed during skin wound healing.¹ MMP-1, -3, -7 and -12 are increased in corneal epithelial cells during Wnt 7a-induced rat cornea wound healing.¹⁶ Wound repair after excimer laser keratectomy is characterized by increased expression of MMP-1, -2, -3 and -9 in the rabbit cornea, and MMP-2, -9 in the rat cornea.^17,18 The expression of MMP-2 and -9 during skin keratinocyte and corneal epithelial cell migration has been the most thoroughly investigated, and it has been shown that their expression generally depends on the activity of MMP-14. MMP-14 (MT1-MMP) is constitutively anchored to the cell membrane; it activates other MMPs such as MMP-2, and also cleaves various types of ECM molecules including collagens, laminins, fibronectin as well as its ligands, the integrins.¹³ The latent forms of some cytokines are also cleaved and activated by MMP-14.¹⁹ Overexpression of MMP-14 protein was found to stimulate HT1080 human fibrosarcoma cell migration.²⁰ In contrast, the attenuation of MMP-14 expression using siRNA method decreased fibroblast invasiveness,²¹ angiogenesis of human microvascular endothelial cells,²² and human skin keratinocyte migration.¹⁰ The latter effect was shown to result from lowering MMP-9 expression. Other studies have shown that EGF has a critical role in MMP-9 expression during keratinocyte tumorigenesis and migration.^23,24 On the other hand, TGFβ modulates MMP-9 production through the Ras/MAPK pathway in transformed mouse keratinocytes and NFκB induces cell migration by binding to the MMP-9 promoter in human skin primary cultures.^25,26 Enhanced levels of pro-MMP-9 and active MMP-9 have also been noted in scratched corneal epithelia of diabetic rats.²⁷There is evidence that MMP-14 activates a number of intracellular signaling pathways including the MAPK family pathway, focal adhesion kinase (FAK), Src family, Rac and CD44, during cell migration and tumor invasion.^19,20,28 In COS-7 cells, ERK activation is stimulated by overexpression of MMP-14 and is essential for cell migration.²⁹ These observations all indicate that MMP-14 plays an important role in cell migration, not only by regulating the activity or expression of downstream MMPs but also by processing and activating migration-associated molecules such as integrins, ECMs and a variety of intracellular signaling pathays.³⁰Cell migration during wound healing is a remarkably complex phenomenon. TGFβ is just one small component of the overall process of wound healing and yet it triggers a multitude of reactions needed for cell migration. It is important to know what kinds of molecules are expressed when cell migration is initiated, but it is equally important to investigate the roles of these molecules and how their expression is regulated. Despite the availability of some information about how MMPs and signaling molecules can influence each other, much remains to be discovered in this area. It will be especially important to clarify how MMP-14 influences other signaling pathways since its role in cell migration is not restricted to digesting ECM molecules but also includes direct or indirect activation of cellular signaling pathways.     

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.     

Regulation of cancer invasiveness by the physical extracellular matrix environment

Long-term clinical outcomes are dependent on whether carcinoma cells leave the primary tumor site and invade through adjacent tissue. Recent evidence links tissue rigidity to alterations in cancer cell phenotype and tumor progression. We found that rigid extracellular matrix (ECM) substrates promote invasiveness of tumor cells via increased activity of invadopodia, subcellular protrusions with associated ECM-degrading proteinases. Although the subcellular mechanism by which substrate rigidity promotes invadopodia function remains to be determined, force sensing does appear to occur through myosin-based contractility and the mechanosensing proteins FAK and p130^Cas. In addition to rigidity, a number of ECM characteristics may regulate the ability of cells to invade through tissues, including matrix density and crosslinking. 3-D biological hydrogels based on type I collagen and reconstituted basement membrane are commonly used to study invasive behavior; however, these models lack some of the tissue-specific properties found in vivo. Thus, new in vitro organotypic and synthetic polymer ECM substrate models will be useful to either mimic the properties of specific ECM microenvironments encountered by invading cancer cells or to manipulate ECM substrate properties and independently test the role of rigidity, integrin ligands, pore size and proteolytic activity in cancer invasion of various tissues.Key words: cancer, invasion, invadopodia, rigidity, mechanotransduction, microenvironmentIn multicellular organisms, cells must sense and respond to multiple cues for proper functioning within tissues. Although most experimental research has focused on the regulation of cellular processes by external chemical signals, there is increasing recognition that mechanical forces also regulate critical cellular functions. Indeed, rigidity of the extracellular environment has been shown to regulate such diverse processes as muscle cell differentiation, stem cell lineage fate, breast epithelial signaling and phenotype, and fibroblast motility.^1–5In breast cancer, accumulating evidence suggests a role for tissue rigidity in promoting both the formation and invasiveness of tumors. Mammographic density of breast tissue has been correlated with increased cancer risk and included in models to predict the likelihood of in situ and invasive breast cancers.⁶ Histologically, dense breast tissue has increased stromal collagen content and in vitro analyses have shown that cancerous breast tissue is much stiffer than normal tissue (as represented by values for the elastic or Young''s moduli).^3,7 In addition, experimentally increased expression of collagen fibrils in a mouse mammary model of spontaneous breast cancer was recently shown to promote tumor formation, invasion and metastasis.⁸ Therefore, both clinical and animal data suggest a correlation between tissue density and cancer aggressiveness, and mechanical factors appear likely to play a role in this process.⁹A well-established mechanism by which extracellular matrix (ECM) rigidity signals can drive phenotypic transformations is through mechano-signal transduction (mechanotransduction) pathways in which external forces are transmitted via integrin receptors at linear focal adhesion structures to cytoskeletal and signaling proteins inside the cell. Actomyosin contractility leads to stretching and activation of proteins such as talin, p130^Cas and potentially focal adhesion kinase (FAK).^10–12 For example, stem cell lineage was found to be dependent on formation of cellular focal adhesions and actomyosin contractility in response to substrate tensile properties.² Mammary epithelial cells grown on compliant matrices will differentiate and polarize to form lactating 3-D structures that resemble in vivo acini but fail to do so on stiff matrices due to increased cytoskeletal contractility.³ Activation of mechanotransduction molecules, such as FAK, Rho and ROCK, are required for the rigidity-induced phenotype changes.^3,5 Using polyacrylamide (PA) gel systems, Yu-li Wang''s group found that rigid substrates induce fibroblast and epithelial cells to migrate away from each other instead of aggregating to form tissue-like structures.¹³ This transformation in phenotype is characteristic of the epithelial to mesenchymal transition and thought to be crucial for tumor cell migration.¹⁴A critical feature of tumor aggressiveness is the ability to invade across tissue boundaries, through degradation of ECM. The subcellular structures responsible for this invasive activity are thought to be invadopodia: actin-rich, finger-like cellular protrusions that proteolytically degrade local ECM. These structures are characteristic of invasive cells and have been implicated in tumor cell metastasis due to their association with ECM degradation.¹⁵ Similar structures, podosomes, are formed in src-transformed cells, as well as normal cells such as osteoclasts and dendritic cells that need to degrade matrix and/or cross tissue boundaries.¹⁶ In addition to mediating ECM degradation, podosomes have been postulated to function as adhesion structures, since well-characterized adhesion proteins localize to podosomes and many podosome-expressing cells no longer express focal adhesions.¹⁷ Furthermore, podosomes have been shown to be essential for chemotactic motility and transendothelial migration, although not for chemokinetic motility.^18,19We recently found that ECM rigidity increases both the number and activity of invadopodia, and this effect was dependent on the cellular contractile machinery (Fig. 1A).²⁰ Consistent with a role for mechanotransduction in this process, we found localization of the active, phosphorylated forms of the mechanosensing proteins FAK and p130^Cas in actively degrading invadopodia and an increase in invadopodia-associated degradation in breast cancer cells overexpressing FAK and p130^Cas. These results suggest that in breast cancer, increases in tissue rigidity may directly lead to increased cellular invasiveness and tumor progression.Open in a separate windowFigure 1Potential rigidity sensing mechanisms by invadopodia. (A) Invadopodia are typically identified by colocalization of fluorescent antibodies for actin and cortactin at puncta that correspond to areas of ECM degradation visualized as dark regions in FITC-labeled fibronectin (Fn) overlaying gelatin. In this case, ECM was layered on top of either soft (storage modulus = 360 Pa) or hard (storage modulus = 3,300 Pa) polyacrylamide gels (PA) to determine if invadopodia activity was regulated by differences in mechanical properties. On hard PA, invasive MCF10ACA1d breast carcinoma cells produced more invadopodia and degraded more ECM than on soft PA. Yellow arrows indicate examples of invadopodia. (B) The localization of rings of the contractile protein myosin IIA (myoIIA) surrounding invadopodia (actin puncta) suggests a role for these structures in mechanosensing by potentially linking invadopodia with the contractile apparatus to detect differences in substrate rigidity. An example ring structure is indicated with a yellow arrow and shown in the zoomed portion of the myosin IIA image, and an example of no or weak localization of myosin IIA with an invadopodium is indicated with the red arrow. (C) Activated forms of FAK and p130^Cas localize to invadopodia and depend on cytoskeletal contractility.²⁰ Rings of myosin IIA also frequently surround invadopodia. These results suggest that invadopodia may act as mechanosensing organelles, either directly through localized mechanoresponsiveness at the invadopodia or through longer-range connections to neighboring or even distant focal adhesions. In either case, traction forces may be generated as a result of changes in cytoskeletal tension in response to ECM properties. Alternatively, invadopodia function could be regulated in the absence of local traction forces, secondary to distant intracellular signaling that leads to alterations in whole cell phenotypic changes. (A and B) are reprinted from Current Biology, Volume 18, Nelson R. Alexander, Kevin M. Branch, Aron Parekh, Emily S. Clark, Izuchukwu C. Iwueke, Scott A. Guelcher and Alissa M. Weaver, Extracellular Matrix Rigidity Promotes Invadopodia Activity, pp. 1295–9, 2008; with permission from Elsevier.The localization of phosphorylated FAK and p130^Cas at invadopodia and the requirement for actomyosin contractility in our study suggests that invadopodia have the potential to act as mechanosensing organelles. This concept is supported by our finding that ∼40% of breast cancer cells cultured on rigid substrates had rings of myosin IIA surrounding invadopodia (Fig. 1B)²⁰ and the recent finding that similar podosome structures can exert local traction forces.²¹ In addition, a few studies have implicated integrin activity in invadopodia function as well as localized β1 and β3 integrins to invadopodia.^22–25 However, whether invadopodia can serve as tension-generating adhesion structures is controversial, in part because of the presence of both focal adhesions and invadopodia in many cancer cells (Fig. 1C).Regulation of invadopodia and podosome function is also not straightforward. Although our data,²⁰ along with results from Collin et al.,²¹ suggests that mechanical tension promotes invadopodia and podosome activity, in some systems podosome formation is promoted by a loss rather than a gain of cytoskeletal tension. That is, local cytoskeletal relaxation has been shown to promote podosome formation coincident with focal adhesion dissolution in both vascular smooth muscle cells treated with phorbol ester²⁶ and neuroblastoma cells.²⁷ A yin-yang activity between focal adhesions and podosomes has been known for many years, whereby activation of src kinase leads to both disassembly of focal adhesions²⁸ and formation of podosomes.²⁹ However, the role of tension in this process is unclear, particularly since activation of src kinase occurs downstream of mechanical stimuli³⁰ and should promote podosome/invadopodia activity, yet loss of tension apparently induces biological activities dependent on src kinase (focal adhesion disassembly and podosome formation).^26,27 For invadopodia, the role of tension is even less clear. Basic characterization studies need to be performed to establish molecular and structural differences between invadopodia and focal adhesions and to measure force profiles at the two structures. Since invadopodia have much smaller diameters compared to podosomes (50–100 nm vs ∼1 µm, respectively),^15,16 the latter task of determining traction forces may be difficult due to resolution limitations in measuring potentially tiny substrate displacements. The standard identification of invadopodia, by association of actin-rich puncta with sites of degradation of fluorescent ECM, adds another technical limitation since the thickness and fluorescence of the ECM matrix used to identify proteolytic activity may hinder visualization of embedded fluorescent beads in the underlying PA gel (displacement of beads is typically used to calculate traction forces).³¹ Thus, an important future direction should be the development of new in vitro experimental systems that have manipulable substrate properties and allow simultaneous identification of subcellular forces and proteolytic activity.The cellular response to rigidity is often characterized using PA gels with tunable stiffness in the range spanning that of normal and cancerous breast tissue (elastic moduli = 100–10,000 Pa).^3,7 PA gels will likely continue to be invaluable tools for understanding cellular responses to rigidity. However, this system is inherently simple and cannot fully replicate cellular events occurring in a complex in vivo ECM microenvironment. Given that invading breast cancer cells are likely to experience different microenvironments as they cross through the basement membrane (BM) and into neighboring collagenous stromal tissue (Fig. 2), biological hydrogels such as reconstituted basement membrane (Matrigel) and type I collagen gels are often utilized to mimic these ECM substrates. However, both of these models lack many of the chemical, physical, and mechanical characteristics of tissues found in vivo and have been recently questioned as suitable models for studying cancer cell invasion.³² Type I collagen gels have a fibrillar architecture but a low density and high porosity³³ and frequently lack crosslinking sites.³⁴ Although Matrigel contains many of the biochemical components of the BM, it is tumor-derived³⁵ and the major component is laminin-1, which is only abundant in fetal tissues.³⁶ By contrast, the major component of normal BM is type IV collagen. In addition, Matrigel is a solubilized preparation that lacks crosslinks³⁷ and a fibrillar component.³⁸ Both sparse collagen gels and Matrigel are quite compliant with Young''s moduli of ∼1,000 and ∼200 Pa, respectively;³ therefore, without further manipulation these substrates lack the rigidity required to mimic tumor-associated ECM.Open in a separate windowFigure 2Navigation of basement membranes and stromal collagen by invading cancer cells. Invasive cancer cells are thought to navigate different tissue microenvironments in the process of invasion. In order for invasion to occur, tumor cells must first breach the basement membrane, a thin and highly crosslinked specialized ECM that requires proteolytic degradation for subsequent transmigration. Once past this barrier, cells must proceed through the neighboring stroma composed of collagenous connective tissue. The meshwork in the stroma is looser and may facilitate diverse migration modes dependent on local microenvironmental conditions and cellular cohesiveness. These modes of migration include a single cell, proteinase-independent amoeboidal phenotype (left) and single cell (middle) and collective (right) proteinase-dependent mesenchymal phenotypes that locally degrade matrix at enzymatically active invadopodia. Note the absence of collagen stroma surrounding and along the migration track of proteolytically active cells. New physiologically relevant models that mimic these interactions in vitro will be useful to elucidate mechanisms of cancer cell migration and invasion in various tissues.In order to invade neighboring stromal tissue, carcinoma cells must first breach the BM, a complex, interwoven meshwork composed of type IV collagen, laminin, nidogen/entactin, and various proteoglycans and glycoproteins.³² The highly ordered and crosslinked type IV collagen network is regarded as the limiting barrier to cancer cell invasion since it forms pores on the order of 100 nm that are too small for passage of cells without proteolytic degradation of the BM.³² In addition to degradation, decreased BM synthesis may contribute to the initial steps of cancer invasion by altering the balance between BM formation and remodeling.³⁹ Once cancer cells cross the BM, they encounter stromal collagen tissue. In tumors, this desmoplastic stroma is frequently fibrotic due to increased ECM deposition and crosslinking by carcinoma-associated fibroblasts.⁹ Although controversial, cancer cells are thought to use a nonproteolytic, amoeboid mode to traverse this connective tissue;⁴⁰ therefore, different modes of migration may be necessary to traverse BM or stromal collagenous matrices (Fig. 2). However, the amoeboid phenotype has been described using either sparse collagen gels without crosslinks⁴¹ or Matrigel.⁴² In vivo, the process of invading through tumor-associated stromal collagen is likely to depend on the pore size, the crosslinking status, and whether cells are migrating collectively or individually.^34,43In light of these concerns and many others, there has been a push for more physiologically relevant in vitro models that represent closer approximations of BM or stromal collagen tissue. Successful models, whether natural or synthetic, must be able to mimic the composition, architecture and mechanical properties of the in vivo environment as well as support cell culture in ex vivo conditions. Natural substrates can be produced by cultured cells, such as the epithelial basement membranes synthesized by MDCK cells.³⁷ Alternatively, organotypic models derived from biological specimens have recently been utilized to study invasion. These materials can be based on processed biological tissue, such as detergent-extracted mouse embryo sections,⁴⁴ homogenized involution matrix,³⁸ and decellularized human dermis,⁴⁵ or on native tissue such as chick chorioallantoic membrane⁴⁶ and explanted peritoneal or mammary tissue.^34,37 In addition, the field of tissue engineering has already provided novel hybrid scaffolds and advanced tissue culturing methods that can be utilized for cancer research.⁴⁷ Biological materials developed for clinical use in tissue reconstruction and regeneration, such as small intestinal submucosa and urinary bladder matrix, are attractive candidates as new in vitro models since they maintain their tissue-like properties and have been extensively characterized.^48,49 These tissue-derived scaffolds are composed of well-defined structural and functional proteins, originally produced by cells in vivo, and maintain their complex 3-D architecture. Thus, such materials can provide an environment that recapitulates the chemical, physical and mechanical properties found in vivo.⁴⁸ In addition, synthetic materials, such as poly(ethylene glycol)-based hydrogels, will likely play a large role in cancer research since they can be designed with defined chemistries to obtain appropriate physical and mechanical properties as well as specific spatial arrangements of biologically relevant moieties on relevant length scales.^33,50 Similarly, engineered adhesive microenvironments created with microfabrication techniques can also be utilized to probe molecular and cellular phenomena.⁵¹ Due to this flexibility in fabrication, these materials are good candidates for novel in vitro models to probe the effects of specific mechanical, topographical and chemical factors on cellular migration and invasion.In summary, the physical microenvironment is increasingly recognized as a major influence on cellular phenotype. Recent data emphasizes the importance of mechanical factors in tumor progression, including cellular invasiveness. Exciting future directions include understanding how stromal and BM environments affect cellular invasiveness at multiple scales, including subcellular and molecular regulation of ECM degradation in response to ECM rigidity and the role of proteinases in crossing diverse tissue barriers. The development of novel model systems with appropriate biological and physical properties will facilitate all of these goals.     

Fasting and differential chemotherapy protection in patients

Chronic calorie restriction has been known for decades to prevent or retard cancer growth, but its weight-loss effect and the potential problems associated with combining it with chemotherapy have prevented its clinical application. Based on the discovery in model organisms that short term starvation (STS or fasting) causes a rapid switch of cells to a protected mode, we described a fasting-based intervention that causes remarkable changes in the levels of glucose, IGF-I and many other proteins and molecules and is capable of protecting mammalian cells and mice from various toxins, including chemotherapy. Because oncogenes prevent the cellular switch to this stress resistance mode, starvation for 48 hours or longer protects normal yeast and mammalian cells and mice but not cancer cells from chemotherapy, an effect we termed Differential Stress Resistance (DSR). In a recent article, ten patients who fasted in combination with chemotherapy, reported that fasting was not only feasible and safe but caused a reduction in a wide range of side effects accompanied by an apparently normal and possibly augmented chemotherapy efficacy. Together with the remarkable results observed in animals, these data provide preliminary evidence in support of the human application of this fundamental biogerontology finding, particularly for terminal patients receiving chemotherapy. Here we briefly discuss the basic, pre-clinical and clinical studies on fasting and cancer therapy.Key words: fasting, cancer, chemotherapy, calorie restriction, stress resistanceAfter decades of slow progress in the identification of treatments effective on a wide range of malignancies, cancer treatment is now turning to personalized therapies based in part on pharmacogenomics. By contrast, aging research is moving in the opposite direction by searching for common ways to prevent, postpone and treat a wide range of age-related diseases, based on the modulation of genetic pathways that are conserved from yeast to mammals.¹ In fact, it may be a solid evolutionary and comparative biology-foundation, which makes this ambitious goal of biogerontologists a realistic or at least a promising one. On the other hand, the progress of biogerontology is viewed by many clinicians as too fundamental and far from translational applications. In most cases, it is not clear how aging research will be translated into FDA approved drugs or treatments that have effects that are superior to those already available or being developed. For example, it is not clear how the long-term 20–30% reduction in calorie intake (dietary restriction, DR) that we and many others before us have shown to be effective in extending the life span of model organisms will make humans live longer or healthier.^1–3 Furthermore, despite the fact that long-term DR was confirmed to reduce cancer and cardiovascular disease in monkeys⁴ and to be effective in preventing obesity, type 2 diabetes, inflammation, hypertension and atherosclerosis, as indicated by the early results in humans studies,⁵ it is highly unlikely to be adopted in its more extreme and effective version by even a small portion of the population. For example, the 20 to 40% chronic reduction in daily calorie intake shown to be effective in retarding cancer growth in mice would not be feasible for cancer therapy for multiple reasons: (1) the effects of chronic DR in patients with a clinically evident tumor is expected to delay but not stop the progression of the disease^6–8 and this delay may only occur for a portion of the malignancies,⁹ (2) although weight loss and cachexia in the early stages of treatment are less prevalent than commonly thought,^10–12 the ∼15% loss of BMI and ∼30% long-term loss of body fat caused by a moderate (20%) calorie restriction¹³ may be tolerated by only a very small portion of cancer patients receiving treatment, (3) Because this long-term restriction is accompanied by delayed wound healing and immunologic impairment in rodents,^1,14,15 it is not clear what risks it may impose on cancer patients receiving treatment.¹⁶ Our studies of DSR, which were triggered by our fundamental findings that switching yeast cells to water protected them against a wide range of toxins, started as a way to address these concerns but also as an attempt to achieve a much more potent therapeutic effect than that achieved by DR.^17,18 Because starvation-induced protection can increase many fold when combined with modulation of pro-aging pathways and since it is in principle blocked by the expression of any oncogene, it has the potential to provide a method to allow common chemotherapy to selectively kill cancer cells, independently of the type of cancer.^19–21 The DSR experiments in mammals were also based on our hypothesis that stress resistance and aging regulatory pathways were conserved from yeast to mammals.We found that fasting for 48 or more hours or in vitro starvation conditions that mimic fasting protected mice and/or normal cells but not cancer cells from various chemotherapy drugs and other deleterious agents.²¹ This effect was shown to depend in part on the reduction of circulating IGF-I and glucose levels.^21,22 Although a differential regulation of cell division in normal and cancer cells^23,24 is likely to contribute to DSR, much of it appears to be dependent on protective systems which are normally maintained in an inactive or low activity state even in non-dividing cells.^1,25 In fact, in non-dividing yeast and mice, deficiencies in glucose or IGF-I signaling that match those observed after starvation promote resistance to doxorubicin, a chemotherapy drug that specifically targets muscle cells in the heart.^21,22As expected, many clinicians were skeptical of our hypothesis that cancer treatment could be improved not by a “magic bullet” but by a “not so magic DSR shield” as underlined by Leonard Saltz, an oncologist at Memorial Sloan-Kettering Cancer Center: “Would I be enthusiastic about enrolling my patients in a trial where they''re asked not to eat for 2.5 days? No.”²⁶ However, ten oncologists did allow their patients, suffering from malignancies ranging from stage II breast cancer to stage IV esophageal, prostate and lung malignancies to undergo a 48–140 hours pre-chemotherapy and a 5–56 hours post chemotherapy water-only fast. The six patients who received chemotherapy with or without fasting reported a reduction in fatigue, weakness and gastrointestinal side effects while fasting²⁷ (Fig. 1). A trend for a reduction of many additional side effects was also reported by the group of patients who always fasted before chemotherapy.²⁷ In those patients whose cancer progression was assessed, chemotherapy was effective and in some cases it was highly effective.²⁷ A clinical trial sponsored by the V-Foundation for Cancer Research, aimed at testing the safety and efficacy of a 24 hour fast in combination with chemotherapy, is in its safety stage. Because it was originally limited to patients diagnosed with bladder cancer the clinical trial progressed slowly. However, its recent expansion to include patients receiving platinum-based chemotherapy (breast, ovarian, lung cancer), is expected to expedite it. Conclusive results for the effect of a 3–4 day fast on chemotherapy-dependent side effects and possibly therapeutic index are not expected to become available for several years. Even if a more modest effect than the 1,000-fold differential protection against oxidative stress and chemotherapy observed in normal and cancer-like yeast cells was achieved in humans, this method could result in long-term survival for many patients with metastatic cancers, particularly those in which malignant cells have not acquired multidrug resistance.Open in a separate windowFigure 1Average self-reported severity of symptoms in patients that have received chemotherapy with or without fasting.     

The roles of cell adhesion molecules in tumor suppression and cell migration: A new paradox

In addition to mediating cell adhesion, many cell adhesion molecules act as tumor suppressors. These proteins are capable of restricting cell growth mainly through contact inhibition. Alterations of these cell adhesion molecules are a common event in cancer. The resulting loss of cell-cell and/or cell-extracellular matrix adhesion promotes cell growth as well as tumor dissemination. Therefore, it is conventionally accepted that cell adhesion molecules that function as tumor suppressors are also involved in limiting tumor cell migration. Paradoxically, in 2005, we identified an immunoglobulin superfamily cell adhesion molecule hepaCAM that is able to suppress cancer cell growth and yet induce migration. Almost concurrently, CEACAM1 was verified to co-function as a tumor suppressor and invasion promoter. To date, the reason and mechanism responsible for this exceptional phenomenon remain unclear. Nevertheless, the emergence of these intriguing cell adhesion molecules with conflicting roles may open a new chapter to the biological significance of cell adhesion molecules.Key words: hepaCAM, cell adhesion molecules, tumor suppressor, migration, E-cadherin, CADM1, integrin α7, CEACAM1It is well known that many cell adhesion molecules function as tumor suppressors (reviewed in ref. ¹). These molecules exert their tumor suppressive effect mainly through cell-adhesion-mediated contact inhibition. Cell adhesion molecules allow cells to communicate with one another or to the extracellular environment by mediating cell-cell or cell-extracellular matrix (ECM) interactions (reviewed in refs. ² and ³). Broadly, these proteins can be classified into five families including immunoglobulin superfamily, integrins, cadherins, selectins and CD44. Apart from participating in the development and maintenance of tissue architecture, cell adhesion molecules serve as cell surface receptors critical for capturing, integrating and transmitting signals from the extracellular milieu to the cell interior (reviewed in refs. ² and ³). These signaling events are vital for the regulation of a wide variety of cellular functions including embryogenesis, immune and inflammatory responses, tissue repair, cell migration, differentiation, proliferation and apoptosis. Alterations of these cell adhesion molecules are a common event in cancer (reviewed in refs. ^{1, 2, 4} and ⁵). The disrupted cell-cell or cell-ECM adhesion significantly contributes to uncontrolled cell proliferation and progressive distortion of normal tissue architecture. More importantly, changes in cell adhesion molecules play a causal role in tumor dissemination. Loss of cell adhesion contacts allows malignant cells to detach and to escape from the primary mass. Gaining a more motile and invasive phenotype, these cells break down the ECM and eventually invade and metastasize to distal organs.Based on the above understanding, it is conventionally accepted that cell adhesion molecules with tumor suppressor activity, when expressed in cancer cells, are able to exert inhibitory effect on cell motility. The ability of cells in migration/motility is a prerequisite for cancer invasion and metastasis (reviewed in refs. ¹ and ⁵). Indeed, a number of cell adhesion molecule-tumor suppressors have been reported to be capable of reducing cell migration. The most classical example is E-cadherin, a calcium-dependent cell adhesion molecule. E-cadherin is expressed exclusively in epithelial cells and its expression is commonly suppressed in tumors of epithelial origins. The cytoplasmic domain of E-cadherin interacts with catenins to establish an intracellular linkage with the actin cytoskeleton (reviewed in ref. ⁶). The assembly of E-cadherin with the cytoskeleton via catenins at the sites of adherens junctions is important for the stabilization of cell-cell adhesions. Disruption of E-cadherin-mediated cell-cell adhesion, due to loss of expression or function of E-cadherin and/or catenins, is assocated with tumor development and progression (reviewed in ref. ⁷). Forced expression of E-cadherin in several cancer cell lines not only slows down cell growth^8,9 but also significantly reduces the invasiveness of the cells.^10,11 On the other hand, inhibition of E-cadherin by function-blocking antibodies and antisense RNA restores the invasiveness in non-invasive transformed cells.¹¹ Furthermore, using a transgenic mouse model of pancreatic beta-cell carcinogenesis, it has been demonstrated that E-cadherin-mediated cell adhesion is important in preventing the transition from well differentiated adenoma to invasive carcinoma.¹²Cell adhesion molecule 1 (CADM1), another example, has also been implicated in cancer progression. CADM1 is a member of the immunoglobulin superfamily and mediates cell-cell adhesion.¹³ The molecule associates with the actin cytoskeleton via the differentially expressed in adenocarcinoma of the lung (DAL1) protein; and the formation of CADM1-DAL1 complex is dependent on the integrity of actin cytoskeleton.¹⁴ Inactivation of the CADM1 and/or DAL1 gene usually through methylation has been reported in diverse human cancers.^15,16 A paper by Ito et al. showed that restoration of CADM1 expression in esophageal squamous cell carcinoma cells not only suppresses cell growth, but also retards cell motility and invasion.¹⁶In contrast to E-cadherin and CADM1, integrin α7 is a cell-ECM adhesion molecule which also possesses tumor suppressor activity. Ren et al. showed that integrin α7 gene is mutated in several human malignances; and the mutations are associated with an increase in cancer recurrence.¹⁷ Forced expression of integrin α7 in integrin α7-deficient leiomyosarcoma cells results in decreased colony formation and slower cell motility. Conversely, knockdown of integrin α7 in lung cancer cells expressing wild-type integrin α7 increases the colony number and cell motility rate. In addition, the researchers revealed that mice bearing xenograft tumors overexpressing integrin α7 have reduced tumor size with no obvious metastasis.In 2005, we first reported the identification of a cell adhesion molecule belonging to the immunoglobulin superfamily, designated as hepaCAM.¹⁸ To date, we have shown that the gene is frequently downregulated in a variety of human cancers.^18,19 Re-expression of hepaCAM in the hepatocellular carcinoma HepG2 cells¹⁸ and breast cancer MCF7 cells¹⁹ inhibits colony formation and retards cell proliferation. In addition, expression of hepaCAM in MCF7 cells results in cell cycle arrest at the G₂/M phase and cellular senescence. Concurrently, the expression of several senescence-associated proteins including p53, p21 and p27 is enhanced. Moreover, downregulation of p53 by p53-specific small interfering RNA in cells expressing hepaCAM clearly reduces p21 without changing p27 and alleviates senescence, indicating that hepaCAM induces senescence through a p53/p21-dependent pathway.¹⁹ Together, the data suggest that hepaCAM is a tumor suppressor. Interestingly, the expression of hepaCAM in both HepG2 and MCF7 cells stimulates both cell-ECM adhesion and cell migration.^18,20,21 The function of hepaCAM as a tumor suppressor in cell migration is contradictory to other cell adhesion molecule-tumor suppressors. Noteworthily, hepaCAM-mediated cell motility is evidenced by its direct interaction with the actin cytoskeleton.²¹Evidences are currently emerging to support the contradictory roles of cell adhesion molecules that both inhibit cell growth and promote cell motility when restored in cancer cells. In addition to hepaCAM, the immunoglobulin superfamily carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is implicated to function as a tumor suppressor and a metastasis promoter. The characteristics and functions of CEACAM1 have been demonstrated in individual reports. CEACAM1 is frequently downregulated or dysregulated in multiple human tumors,^22–25 and is capable of suppressing cell growth and inducing apoptosis.^26–28 Ebrahimnejad et al. demonstrated that exogenous expression of CEACAM1 enhances melanoma cell invasion and migration; and this enhanced motility can be reverted by anti-CEACAM antibodies.²⁹ The ability of CEACAM to co-stimulate tumor suppression and invasion was finally established by Liu et al. in restricting thyroid cancer growth but promoting invasiveness.³⁰ Introduction of CEACAM1 into CEACAM1-deficient thyroid cancer cells results in G₁/S phase cell cycle arrest accompanied by elevated p21 expression and diminished Rb phosphorylation. Overexpression of CEACAM1 also increases cell-ECM adhesion and promotes cell migration and tumor invasiveness. In xenografted mice, CEACAM1 expression results in reduced tumor growth but increased tumor invasiveness. Conversely, silencing of endogenous CEACAM1 accelerates tumor growth and suppresses invasiveness.³⁰It is an exciting issue to address why a cell adhesion molecule is able to suppress tumor growth yet promote tumor progression. Could there be a molecular switch that controls the functions of the gene between a tumor suppressor and a migration promoter in cancer or are the functions executed simultaneously? The expression level, the extracellular cues as well as the interacting partners of the cell adhesion molecules may likely play a critical role in regulating its functions. The question is under what circumstances these factors come into play. To answer all these questions, and maybe more, on the intriguing findings of these proteins, more extensive and intensive experimentation is required. Nevertheless, it is obvious that the emergence of these cell adhesion molecules that function in a contradictory manner opens a new chapter to the biological significance of cell adhesion molecules.     

Human non-CG methylation: Are human stem cells plant-like?

Non-CG methylation is well characterized in plants where it appears to play a role in gene silencing and genomic imprinting. Although strong evidence for the presence of non-CG methylation in mammals has been available for some time, both its origin and function remain elusive. In this review we discuss available evidence on non-CG methylation in mammals in light of evidence suggesting that the human stem cell methylome contains significant levels of methylation outside the CG site.Key words: non-CG methylation, stem cells, Dnmt1, Dnmt3a, human methylomeIn plant cells non-CG sites are methylated de novo by Chromomethylase 3, DRM1 and DRM2. Chromomethylase 3, along with DRM1 and DRM2 combine in the maintenance of methylation at symmetric CpHpG as well as asymmetric DNA sites where they appear to prevent reactivation of transposons.¹ DRM1 and DRM2 modify DNA de novo primarily at asymmetric CpH and CpHpH sequences targeted by siRNA.²Much less information is available on non-CG methylation in mammals. In fact, studies on mammalian non-CG methylation form a tiny fraction of those on CG methylation, even though data for cytosine methylation in other dinucleotides, CA, CT and CC, have been available since the late 1980s.³ Strong evidence for non-CG methylation was found by examining either exogenous DNA sequences, such as plasmid and viral integrants in mouse and human cell lines,^4,5 or transposons and repetitive sequences such as the human L1 retrotransposon⁶ in a human embryonic fibroblast cell line. In the latter study, non-CG methylation observed in L1 was found to be consistent with the capacity of Dnmt1 to methylate slippage intermediates de novo.⁶Non-CG methylation has also been reported at origins of replication^7,8 and a region of the human myogenic gene Myf3.⁹ The Myf3 gene is silenced in non-muscle cell lines but it is not methylated at CGs. Instead, it carries several methylated cytosines within the sequence CCTGG. Gene-specific non-CG methylation was also reported in a study of lymphoma and myeloma cell lines not expressing many B lineage-specific genes.¹⁰ The study focused on one specific gene, B29 and found heavy CG promoter methylation of that gene in most cell lines not expressing it. However, in two other cell lines where the gene was silenced, cytosine methylation was found almost exclusively at CCWGG sites. The authors provided evidence suggesting that CCWGG methylation was sufficient for silencing the B29 promoter and that methylated probes based on B29 sequences had unique gel shift patterns compared to non-methylated but otherwise identical sequences.¹⁰ The latter finding suggests that the presence of the non-CG methylation causes changes in the proteins able to bind the promoter, which could be mechanistically related to the silencing seen with this alternate methylation.Non-CG methylation is rarely seen in DNA isolated from cancer patients. However, the p16 promoter region was reported to contain both CG and non-CG methylation in breast tumor specimens but lacked methylation at these sites in normal breast tissue obtained at mammoplasty.¹¹ Moreover, CWG methylation at the CCWGG sites in the calcitonin gene is not found in normal or leukemic lymphocyte DNA obtained from patients.¹² Further, in DNA obtained from breast cancer patients, MspI sites that are refractory to digestion by MspI and thus candidates for CHG methylation were found to carry CpG methylation.¹³ Their resistance to MspI restriction was found to be caused by an unusual secondary structure in the DNA spanning the MspI site that prevents restriction.¹³ This latter observation suggests caution in interpreting EcoRII/BstNI or EcoRII/BstOI restriction differences as due to CWG methylation, since in contrast to the 37°C incubation temperature required for full EcoRII activity, BstNI and BstOI require incubation at 60°C for full activity where many secondary structures are unstable.The recent report by Lister et al.¹⁴ confirmed a much earlier report by Ramsahoye et al.¹⁵ suggesting that non-CG methylation is prevalent in mammalian stem cell lines. Nearest neighbor analysis was used to detect non-CG methylation in the earlier study on the mouse embryonic stem (ES) cell line,¹⁵ thus global methylation patterning was assessed. Lister et al.¹⁴ extend these findings to human stem cell lines at single-base resolution with whole-genome bisulfite sequencing. They report¹⁴ that the methylome of the human H1 stem cell line and the methylome of the induced pluripotent IMR90 (iPS) cell line are stippled with non-CG methylation while that of the human IMR90 fetal fibroblast cell line is not. While the results of the two studies are complementary, the human methylome study addresses locus specific non-CG methylation. Based on that data,¹⁴ one must conclude that non-CG methylation is not carefully maintained at a given site in the human H1 cell line. The average non-CG site is picked up as methylated in about 25% of the reads whereas the average CG methylation site is picked up in 92% of the reads. Moreover, non-CG methylation is not generally present on both strands and is concentrated in the body of actively transcribed genes.¹⁴Even so, the consistent finding that non-CG methylation appears to be confined to stem cell lines,^14,15 raises the possibility that cancer stem cells¹⁶ carry non-CG methylation while their nonstem progeny in the tumor carry only CG methylation. Given the expected paucity of cancer stem cells in a tumor cell population, it is unlikely that bisulfite sequencing would detect non-CG methylation in DNA isolated from tumor cells since the stem cell population is expected to be only a very minor component of tumor DNA. Published sequences obtained by bisulfite sequencing generally report only CG methylation, and to the best of our knowledge bisulfite sequenced tumor DNA specimens have not reported non-CG methylation. On the other hand, when sequences from cell lines have been reported, bisulfite-mediated genomic sequencing⁸ or ligation mediated PCR¹⁷ methylcytosine signals outside the CG site have been observed. In a more recent study plasmid DNAs carrying the Bcl2-major breakpoint cluster¹⁸ or human breast cancer DNA¹³ treated with bisulfite under non-denaturing conditions, cytosines outside the CG side were only partially converted on only one strand¹⁸ or at a symmetrical CWG site.¹³ In the breast cancer DNA study the apparent CWG methylation was not detected when the DNA was fully denatured before bisulfite treatment.¹³In both stem cell studies, non-CG methylation was attributed to the Dnmt3a,^14,15 a DNA methyltransferase with similarities to the plant DRM methyltransferase family¹⁹ and having the capacity to methylate non-CG sites when expressed in Drosophila melanogaster.¹⁵ DRM proteins however, possess a unique permuted domain structure found exclusively in plants¹⁹ and the associated RNA-directed non-CG DNA methylation has not been reproducibly observed in mammals despite considerable published^20–23 and unpublished efforts in that area. Moreover, reports where methylation was studied often infer methylation changes from 5AzaC reactivation studies²⁴ or find that CG methylation seen in plants but not non-CG methylation is detected.^21,22,25,26 In this regard, it is of interest that the level of non-CG methylation reported in stem cells corresponds to background non-CG methylation observed in vitro with human DNA methyltransferase I,²⁷ and is consistent with the recent report that cultured stem cells are epigenetically unstable.²⁸The function of non-CG methylation remains elusive. A role in gene expression has not been ruled out, as the studies above on Myf3 and B29 suggest.^9,10 However, transgene expression of the bacterial methyltransferase M.EcoRII in a human cell line (HK293), did not affect the CG methylation state at the APC and SerpinB5 genes²⁹ even though the promoters were symmetrically de novo methylated at ^mCWGs within each CCWGG sequence in each promoter. This demonstrated that CG and non-CG methylation are not mutually exclusive as had been suggested by earlier reports.^9,10 That observation is now extended to the human stem cell line methylome where CG and non-CG methylation co-exist.¹⁴ Gene expression at the APC locus was likewise unaffected by transgene expression of M.EcoRII. In those experiments genome wide methylation of the CCWGG site was detected by restriction analysis and bisulfite sequencing,²⁹ however stem cell characteristics were not studied.Many alternative functions can be envisioned for non-CG methylation, but the existing data now constrains them to functions that involve low levels of methylation that are primarily asymmetric. Moreover, inheritance of such methylation patterns requires low fidelity methylation. If methylation were maintained with high fidelity at particular CHG sites one would expect that the spontaneous deamination of 5-methylcytosine would diminish the number of such sites, so as to confine the remaining sites to those positions performing an essential function, as is seen in CG methylation.^30–33 However, depletion of CWG sites is not observed in the human genome.³⁴ Since CWG sites account for only about 50% of the non-CG methylation observed in the stem cell methylome¹⁴ where methylated non-CG sites carry only about 25% methylation, the probability of deamination would be about 13% of that for CWG sites that are subject to maintenance methylation in the germ line. Since mutational depletion of methylated cytosines has to have its primary effect on the germ line, if the maintenance of non-CG methylation were more accurate and more widespread, one would have had to argue that stem cells in the human germ lines lack CWG methylation. As it is the data suggests that whatever function non-CG methylation may have in stem cells, it does not involve accurate somatic inheritance in the germ line.The extensive detail on non-CG methylation in the H1 methylome¹⁴ raises interesting questions about the nature of this form of methylation in human cell lines. A key finding in this report is the contrast between the presence of non-CG methylation in the H1 stem cell line and its absence in the IMR90 human fetal lung fibroblast cell line.¹⁴ This suggests that it may have a role in the origin and maintenance of the pluripotent lineage.¹⁴By analogy with the well known methylated DNA binding proteins specific for CG methylation,³⁵ methylated DNA binding proteins that selectively bind sites of non-CG methylation are expected to exist in stem cells. Currently the only protein reported to have this binding specificity is human Dnmt1.^36–38 While Dnmt1 has been proposed to function stoichiometrically³⁹ and could serve a non-CG binding role in stem cells, this possibility and the possibility that other stem-cell specific non-CG binding proteins might exist remain to be been explored.Finally, the nature of the non-CG methylation patterns in human stem cell lines present potentially difficult technical problems in methylation analysis. First, based on the data in the H1 stem cell methylome,⁴⁰ a standard MS-qPCR for non-CG methylation would be impractical because non-CG sites are infrequent, rarely clustered and are generally characterized by partial asymmetric methylation. This means that a PCR primer that senses the 3 adjacent methylation sites usually recommended for MS-qPCR primer design^41,42 cannot be reliably found. For example in the region near Oct4 (Chr6:31,246,431), a potential MS-qPCR site exists with a suboptimal set of two adjacent CHG sites both methylated on the + strand at Chr6:31,252,225 and 31,252,237.^14,40 However these sites were methylated only in 13/45 and 30/52 reads. Thus the probability that they would both be methylated on the same strand is about 17%. Moreover, reverse primer locations containing non-CG methylation sites are generally too far away for practical bisulfite mediated PCR. Considering the losses associated with bisulfite mediated PCR⁴³ the likelihood that such an MS-qPCR system would detect non-CG methylation in the H1 cell line or stem cells present in a cancer stem cell niche^44,45 is very low.The second difficulty is that methods based on the specificity of MeCP2 and similar methylated DNA binding proteins for enriching methylated DNA (e.g., MIRA,⁴⁶ COMPARE-MS⁴⁷) will discard sequences containing non-CG methylation since they require cooperative binding afforded by runs of adjacent methylated CG sites for DNA capture. This latter property of the methylated cytosine capture techniques makes it also unlikely that methods based on 5-methylcytosine antibodies (e.g., meDIP⁴⁸) will capture non-CG methylation patterns accurately since the stem cell methylome shows that adjacent methylated non-CG sites are rare in comparison to methylated CG sites.¹⁴In summary, whether or not mammalian stem cells in general or human stem cells in particular possess functional plant-like methylation patterns is likely to continue to be an interesting and challenging question. At this point we can conclude that the non-CG patterns reported in human cells appear to differ significantly from the non-CG patterns seen in plants, suggesting that they do not have a common origin or function.     

Cell Migration from Baby to Mother

Fetal cells migrate into the mother during pregnancy. Fetomaternal transfer probably occurs in all pregnancies and in humans the fetal cells can persist for decades. Microchimeric fetal cells are found in various maternal tissues and organs including blood, bone marrow, skin and liver. In mice, fetal cells have also been found in the brain. The fetal cells also appear to target sites of injury. Fetomaternal microchimerism may have important implications for the immune status of women, influencing autoimmunity and tolerance to transplants. Further understanding of the ability of fetal cells to cross both the placental and blood-brain barriers, to migrate into diverse tissues, and to differentiate into multiple cell types may also advance strategies for intravenous transplantation of stem cells for cytotherapeutic repair. Here we discuss hypotheses for how fetal cells cross the placental and blood-brain barriers and the persistence and distribution of fetal cells in the mother.Key Words: fetomaternal microchimerism, stem cells, progenitor cells, placental barrier, blood-brain barrier, adhesion, migrationMicrochimerism is the presence of a small population of genetically distinct and separately derived cells within an individual. This commonly occurs following transfusion or transplantation.^1–3 Microchimerism can also occur between mother and fetus. Small numbers of cells traffic across the placenta during pregnancy. This exchange occurs both from the fetus to the mother (fetomaternal)^4–7 and from the mother to the fetus.^8–10 Similar exchange may also occur between monochorionic twins in utero.^11–13 There is increasing evidence that fetomaternal microchimerism persists lifelong in many child-bearing women.^7,14 The significance of fetomaternal microchimerism remains unclear. It could be that fetomaternal microchimerism is an epiphenomenon of pregnancy. Alternatively, it could be a mechanism by which the fetus ensures maternal fitness in order to enhance its own chances of survival. In either case, the occurrence of pregnancy-acquired microchimerism in women may have implications for graft survival and autoimmunity. More detailed understanding of the biology of microchimeric fetal cells may also advance progress towards cytotherapeutic repair via intravenous transplantation of stem or progenitor cells.Trophoblasts were the first zygote-derived cell type found to cross into the mother. In 1893, Schmorl reported the appearance of trophoblasts in the maternal pulmonary vasculature.¹⁵ Later, trophoblasts were also observed in the maternal circulation.^16–20 Subsequently various other fetal cell types derived from fetal blood were also found in the maternal circulation.^21,22 These fetal cell types included lymphocytes,²³ erythroblasts or nucleated red blood cells,^24,25 haematopoietic progenitors^7,26,27 and putative mesenchymal progenitors.^14,28 While it has been suggested that small numbers of fetal cells traffic across the placenta in every human pregnancy,^29–31 trophoblast release does not appear to occur in all pregnancies.³² Likewise, in mice, fetal cells have also been reported in maternal blood.^33,34 In the mouse, fetomaternal transfer also appears to occur during all pregnancies.³⁵     

Serine/threonine protein phosphatases: Multi-purpose enzymes in control of defense mechanisms

Depending on the threat to a plant, different pattern recognition receptors, such as receptor-like kinases, identify the stress and trigger action by appropriate defense response development.^1,2 The plant immunity system primary response to these challenges is rapid accumulation of phytohormones, such as ethylene (ET), salicylic acid (SA), and jasmonic acid (JA) and its derivatives. These phytohormones induce further signal transduction and appropriate defenses against biotic threats.^3,4 Phytohormones play crucial roles not only in the initiation of diverse downstream signaling events in plant defense but also in the activation of effective defenses through an essential process called signaling pathway crosstalk, a mechanism involved in transduction signals between two or more distinct, “linear signal transduction pathways simultaneously activated in the same cell.”⁵     

Neutralization of Clostridium difficile toxin A using antibody combinations

The pathogenicity of Clostridium difficile (C. difficile) is mediated by the release of two toxins, A and B. Both toxins contain large clusters of repeats known as cell wall binding (CWB) domains responsible for binding epithelial cell surfaces. Several murine monoclonal antibodies were generated against the CWB domain of toxin A and screened for their ability to neutralize the toxin individually and in combination. Three antibodies capable of neutralizing toxin A all recognized multiple sites on toxin A, suggesting that the extent of surface coverage may contribute to neutralization. Combination of two noncompeting antibodies, denoted 3358 and 3359, enhanced toxin A neutralization over saturating levels of single antibodies. Antibody 3358 increased the level of detectable CWB domain on the surface of cells, while 3359 inhibited CWB domain cell surface association. These results suggest that antibody combinations that cover a broader epitope space on the CWB repeat domains of toxin A (and potentially toxin B) and utilize multiple mechanisms to reduce toxin internalization may provide enhanced protection against C. difficile-associated diarrhea.Key words: Clostridium difficile, toxin neutralization, therapeutic antibody, cell wall binding domains, repeat proteins, CROPs, mAb combinationThe most common cause of nosocomial antibiotic-associated diarrhea is the gram-positive, spore-forming anaerobic bacillus Clostridium difficile (C. difficile). Infection can be asymptomatic or lead to acute diarrhea, colitis, and in severe instances, pseudomembranous colitis and toxic megacolon.^1,2The pathological effects of C. difficile have long been linked to two secreted toxins, A and B.^3,4 Some strains, particularly the virulent and antibiotic-resistant strain 027 with toxinotype III, also produce a binary toxin whose significance in the pathogenicity and severity of disease is still unclear.⁵ Early studies including in vitro cell-killing assays and ex vivo models indicated that toxin A is more toxigenic than toxin B; however, recent gene manipulation studies and the emergence of virulent C. difficile strains that do not express significant levels of toxin A (termed “A⁻ B⁺”) suggest a critical role for toxin B in pathogenicity.^6,7Toxins A and B are large multidomain proteins with high homology to one another. The N-terminal region of both toxins enzymatically glucosylates small GTP binding proteins including Rho, Rac and CDC42,^8,9 leading to altered actin expression and the disruption of cytoskeletal integrity.^9,10 The C-terminal region of both toxins is composed of 20 to 30 residue repeats known as the clostridial repetitive oligopeptides (CROPs) or cell wall binding (CWB) domains due to their homology to the repeats of Streptococcus pneumoniae LytA,^11–14 and is responsible for cell surface recognition and endocytosis.^12,15–17C. difficile-associated diarrhea is often, but not always, induced by antibiotic clearance of the normal intestinal flora followed by mucosal C. difficile colonization resulting from preexisting antibiotic resistant C. difficile or concomitant exposure to C. difficile spores, particularly in hospitals. Treatments for C. difficile include administration of metronidazole or vancomycin.^2,18 These agents are effective; however, approximately 20% of patients relapse. Resistance of C. difficile to these antibiotics is also an emerging issue^19,20 and various non-antibiotic treatments are under investigation.^20–25Because hospital patients who contract C. difficile and remain asymptomatic have generally mounted strong antibody responses to the toxins,^26,27 active or passive immunization approaches are considered hopeful avenues of treatment for the disease. Toxins A and B have been the primary targets for immunization approaches.^20,28–33 Polyclonal antibodies against toxins A and B, particularly those that recognize the CWB domains, have been shown to effectively neutralize the toxins and inhibit morbidity in rodent infection models.³¹ Monoclonal antibodies (mAbs) against the CWB domains of the toxins have also demonstrated neutralizing capabilities; however, their activity in cell-based assays is significantly weaker than that observed for polyclonal antibody mixtures.^33–36We investigated the possibility of creating a cocktail of two or more neutralizing mAbs that target the CWB domain of toxin A with the goal of synthetically re-creating the superior neutralization properties of polyclonal antibody mixtures. Using the entire CWB domain of toxin A, antibodies were raised in rodents and screened for their ability to neutralize toxin A in a cell-based assay. Two mAbs, 3358 and 3359, that (1) both independently demonstrated marginal neutralization behavior and (2) did not cross-block one another from binding toxin A were identified. We report here that 3358 and 3359 use differing mechanisms to modify CWB-domain association with CHO cell surfaces and combine favorably to reduce toxin A-mediated cell lysis.     

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.