Podosomes and Invadopodia: Related structures with Common Protein Components that May Promote Breast Cancer Cellular Invasion

A rate-limiting step in breast cancer progression is acquisition of the invasive phenotype, which can precede metastasis. Expression of cell-surface proteases at the leading edge of a migrating cell provides cells with a mechanism to cross tissue barriers. A newly appreciated mechanism that may be relevant for breast cancer cell invasion is the formation of invadopodia, well-defined structures that project from the ventral membrane and promote degradation of the extracellular matrix, allowing the cell to cross a tissue barrier. Recently, there has been some controversy and discussion as to whether invadopodia, which are associated with carcinoma cells, are related to a similar structure called podosomes, which are associated with normal cells. Invadopodia and podosomes share many common characteristics, including a similar size, shape, subcellular localization and an ability to promote invasion. These two structures also share many common protein components, which we outline herein. It has been speculated that podosomes may be precursors to invadopodia and by extension both structures may be relevant to cancer cell invasion. Here, we compare and contrast the protein components of invadopodia and podosomes and discuss a potential role for these proteins and the evidence that supports a role for invadopodia and podosomes in breast cancer invasion

A Polymorphic Variant of AFAP-110 Enhances cSrc Activity12

Enhanced expression and activity of cSrc are associated with ovarian cancer progression. Generally, cSrc does not contain acti- vating mutations; rather, its activity is increased in response to signals that affect a conformational change that releases its auto- inhibition. In this report, we analyzed ovarian cancer tissues for the expression of a cSrc-activating protein, AFAP-110. AFAP-110 activates cSrc through a direct interaction that releases it from its autoinhibited conformation. Immunohistochemical analysis re- vealed a concomitant increase of AFAP-110 and cSrc in ovarian cancer tissues. An analysis of the AFAP-110 coding sequence revealed the presence of a nonsynonymous, single-nucleotide polymorphism that resulted in a change of Ser403 to Cys403. In cells that express enhanced levels of cSrc, AFAP-110403C directed the activation of cSrc and the formation of podosomes indepen- dently of input signals, in contrast to wild-type AFAP-110. We therefore propose that, under conditions of cSrc overexpression, the polymorphic variant of AFAP-110 promotes cSrc activation. Further, these data indicate a mechanism by which an inherited genetic variation could influence ovarian cancer progression and could be used to predict the response to targeted therapy

AFAP1 Is a Novel Downstream Mediator of TGF-β1 for CCN2 Induction in Osteoblasts.

BACKGROUND:CCN2 acts as an anabolic growth factor to regulate osteoblast differentiation and function. CCN2 is induced by TGF-β1 and acts as a mediator of TGF-β1 induced matrix production in osteoblasts and Src is required for CCN2 induction by TGF-β1; however, the molecular mechanisms that control CCN2 induction in osteoblasts are poorly understood. AFAP1 binds activated forms of Src and can direct the activation of Src in certain cell types, however a role for AFAP1 downstream of TGF-β1 or in osteoblats is undefined. In this study, we investigated the role of AFAP1 for CCN2 induction by TGF-β1 in primary osteoblasts. RESULTS:We demonstrated that AFAP1 expression in osteoblasts occurs in a biphasic pattern with maximal expression levels occurring during osteoblast proliferation (~day 3), reduced expression during matrix production/maturation (~day 14-21), an a further increase in expression during mineralization (~day 21). AFAP1 expression is induced by TGF-β1 treatment in osteoblasts during days 7, 14 and 21. In osteoblasts, AFAP1 binds to Src and is required for Src activation by TGF-β1 and CCN2 promoter activity and protein induction by TGF-β1 treatment was impaired using AFAP1 siRNA, indicating the requirement of AFAP1 for CCN2 induction by TGF-β1. We also demonstrated that TGF-β1 induction of extracellular matrix protein collagen XIIa occurs in an AFAP1 dependent fashion. CONCLUSIONS:This study demonstrates that AFAP1 is an essential downstream signaling component of TGF-β1 for Src activation, CCN2 induction and collagen XIIa in osteoblasts

Bcr and Abr Cooperate in Negatively Regulating Acute Inflammatory Responses

Bcr and Abr are GTPase-activating proteins for the small GTPase Rac. Both proteins are expressed in cells of the innate immune system, including neutrophils and macrophages. The function of Bcr has been linked to the negative regulation of neutrophil reactive oxygen species (ROS) production, but the function of Abr in the innate immune system was unknown. Here, we report that mice lacking both proteins are severely affected in two models of experimental endotoxemia, including exposure to Escherichia coli lipopolysaccharide and polymicrobial sepsis, with extensive microvascular leakage, resulting in severe pulmonary edema and hemorrhage. Additionally, in vivo-activated neutrophils of abr and bcr null mutant mice produced excessive tissue-damaging myeloperoxidase (MPO), elastase, and ROS. Moreover, the secretion of the tissue metalloproteinase MMP9 by monocytes and ROS by elicited macrophages was abnormally high. In comparison, ROS production from bone marrow monocytes was not significantly different from that of controls, and the exocytosis of neutrophil secondary and tertiary granule products, including lactoferrin, was normal. These data show that Abr and Bcr normally curb very specific functions of mature tissue innate immune cells, and that each protein has distinct as well as partly overlapping functions in the downregulation of inflammatory processes

AFAP1 is necessary for TGF-β1 induced Col XIIa.

<p>The data show that blocking AFAP1 expression with siRNA impairs Col XIIa protein expression. Primary osteoblasts were pretreated with AFAP1 siRNA (siRNA) and then treated with TGF-β1 (5ng/ml; 24hrs) (+) or mock treated (-) and Col XIIa, AFAP1, CCN2 and Actin were assessed by Western Blot. Western Blots are representative of triplicate determinations.</p

AFAP1 is required for CCN2 induction by TGF-β1.

<p>The data show that blocking AFAP1 expression with siRNA impairs CCN2 protein and promoter induction by TGF-β1 in primary osteoblasts. Additionally, CCN2 induction by TGF-β1 is impaired in osteoblasts derived from AFAP1^-/- mice. <u>Methods:</u><b>(A)</b> Primary osteoblasts were pretreated with AFAP1 siRNA (siRNA) or non-targeting control siRNA (control) or transfection reagent only (transfectant only) and then treated with TGF-β1 (5ng/ml; 24hrs) (+) or mock treated (-) and AFAP1, CCN2 and Actin were assessed by Western blot. (<b>B</b>) Primary Osteoblasts were treated as in A, co-transfected with a full length CCN2 proximal promoter reporter, serum starved and then treated with TGF-β1 (5ng/ml) for 24hrs. Luciferase is expressed as a ratio of firefly/renilla. (SEM, n = 6) star symbol = p<0.05 compared to TGF-B1 +, control siRNA sample.</p