Angiosarcoma (AS) is a rare neoplasm of endothelial origin that has limited treatment options and poor five-year survival. As a model for human AS, we studied primary cells and tumorgrafts derived from canine hemangiosarcoma (HSA), which is also an endothelial malignancy with similar presentation and histology. Primary cells isolated from HSA showed constitutive ERK activation. The MEK inhibitor CI-1040 reduced ERK activation and the viability of primary cells derived from visceral, cutaneous, and cardiac HSA in vitro. HSA-derived primary cells were also sensitive to sorafenib, an inhibitor of B-Raf and multi-receptor tyrosine kinases. In vivo, CI-1040 or PD0325901 decreased the growth of cutaneous cell-derived xenografts and cardiac-derived tumorgrafts. Sorafenib decreased tumor size in both in vivo models, although cardiac tumorgrafts were more sensitive. In human AS, we noted that 50% of tumors stained positively for phosphorylated ERK1/2 and that the expression of several MEK-responsive transcription factors was up-regulated. Our data showed that MEK signaling is essential for the growth of HSA in vitro and in vivo and provided evidence that the same pathways are activated in human AS. This indicates that MEK inhibitors may form part of an effective therapeutic strategy for the treatment of canine HSA or human AS, and it highlights the utility of spontaneous canine cancers as a model of human disease.
Hemangiosarcoma; Angiosarcoma; MEK; ERK; CI-1040
Infection by Bacillus anthracis in animals and humans results from accidental or intentional exposure, by oral, cutaneous or pulmonary routes, to spores, which are normally present in the soil. Treatment includes administration of antibiotics, vaccination or treatment with antibody to the toxin. A better understanding of the molecular basis of the processes involved in the pathogenesis of anthrax namely, spore germination in macrophages and biological effects of the secreted toxins on heart and blood vessels will lead to improved management of infected animals and patients. Controlling germination will be feasible by inhibiting macrophage paralysis and cell death. On the other hand, the control of terminal hypotension might be achieved by inhibition of cardiomyocyte mitogen-activated protein kinase and stimulation of vessel cAMP.
Anthrax; Heart Failure; Rats; Review
Human anthrax infection caused by exposure to Bacillus anthracis cannot always be treated by antibiotics. This is mostly because of the effect of the remaining anthrax toxin in the body. Lethal factor (LF) is a component of lethal toxin (LeTx), which is the major virulence of anthrax toxin. A murine IgG monoclonal antibody (mAb) against LF with blocking activity (coded LF8) was produced in a previous study. In this report, a human/murine chimeric Fab mAb (coded LF8-Fab) was developed from LF8 by inserting murine variable regions into human constant regions using antibody engineering to reduce the incompatibility of the murine antibody for human use. The LF8-Fab expressed in Escherichia coli could specifically identify LF with an affinity of 3.46 × 107 L/mol and could neutralize LeTx with an EC50 of 85 μg/mL. Even after LeTx challenge at various time points, the LF8-Fab demonstrated protection of J774A.1 cells in vitro. The results suggest that the LF8-Fab might be further characterized and potentially be used for clinical applications against anthrax infection.
The mitogen-activated protein kinase kinases (the MAPK/ERK kinases; MKKs or MEKs) and their downstream substrates, the extracellular-regulated kinases have been intensively studied for their roles in development and disease. Until recently, it had been assumed any mutation affecting their function would have lethal consequences. However, the identification of MEK1 and MEK2 mutations in developmental syndromes as well as chemotherapy-resistant tumors, and the discovery of genomic variants in MEK1 and MEK2 have led to the realization the extent of genomic variation associated with MEKs is much greater than had been appreciated. In this review, we will discuss these recent advances, relating them to what is currently understood about the structure and function of MEKs, and describe how they change our understanding of the role of MEKs in development and disease.
MEK; MAPK; ERK; cardio-facial cutaneous syndrome; cancer; SNP
Mitogen-activated protein kinase kinases (MKK or MEK) 1 and 2 are usually treated as redundant kinases. However, in assessing their relative contribution towards ERK-mediated biologic response investigators have relied on tests of necessity, not sufficiency. In response we developed a novel experimental model using lethal toxin (LeTx), an anthrax toxin-derived pan-MKK protease, and genetically engineered protease resistant MKK mutants (MKKcr) to test the sufficiency of MEK signaling in melanoma SK-MEL-28 cells. Surprisingly, ERK activity persisted in LeTx-treated cells expressing MEK2cr but not MEK1cr. Microarray analysis revealed non-overlapping downstream transcriptional targets of MEK1 and MEK2, and indicated a substantial rescue effect of MEK2cr on proliferation pathways. Furthermore, LeTx efficiently inhibited the cell proliferation and anchorage-independent growth of SK-MEL-28 cells expressing MKK1cr but not MEK2cr. These results indicate in SK-MEL-28 cells MEK1 and MEK2 signaling pathways are not redundant and interchangeable for cell proliferation. We conclude that in the absence of other MKK, MEK2 is sufficient for SK-MEL-28 cell proliferation. MEK1 conditionally compensates for loss of MEK2 only in the presence of other MKK.
Patients with anaplastic thyroid carcinoma (ATC) typically succumb to their disease months after diagnosis despite aggressive therapy. A large percentage of ATCs have been shown to harbor the V600E B-Raf point mutation, leading to the constitutive activation of the mitogen-activated protein kinase (MAPK) pathway. ATC invasion, metastasis, and angiogenesis are in part dependent on the gelatinase class of matrix metalloproteinases (MMPs). The explicit targeting of these two tumor markers may provide a novel therapeutic strategy for the treatment of ATC. The MMP-activated Anthrax Lethal Toxin (LeTx), a novel recombinant protein toxin combination, demonstrates potent MAPK pathway inhibition in gelatinase-expressing V600E B-Raf tumor cells in vitro. However, preliminary in vivo studies showed that the MMP-activated LeTx also exhibited dramatic anti-tumor activity against xenografts that did not show significant anti-proliferative responses to the LeTx in vitro. Here we show that the MMP-activated LeTx inhibits orthotopic ATC xenograft progression in both toxin-sensitive and resistant ATC cells via reduced endothelial cell recruitment and subsequent tumor vascularization. This in turn translates to an improved long-term survival that is comparable to that produced by the multi-kinase inhibitor sorafenib. Our results also indicate that therapy with the MMP-activated LeTx is extremely effective against advanced tumors with well-established vascular networks. Taken together, these results suggest that the MMP-activated LeTx-mediated endothelial cell targeting is the primary in vivo anti-tumor mechanism of this novel toxin. Therefore, the MMP-activated LeTx could be used not only in the clinical management of V600E B-Raf ATC, but potentially in any solid tumor.
Anaplastic thyroid carcinoma; anthrax lethal toxin; lethal factor; matrix metalloproteinase; protective antigen; tumor angiogenesis; vascular endothelial growth factor
Anthrax is caused by the gram-positive bacterium Bacillus anthracis. The pathogenesis of this disease is dependent on the presence of two binary toxins, edema toxin (EdTx) and lethal toxin (LeTx). LeTx, the major virulence factor contributing to anthrax, contains the effector moiety lethal factor (LF), a zinc-dependent metalloprotease specific for targeting mitogen-activated protein kinase kinases. This review will focus on the protease-specific activity and function of LF, and will include a discussion on the implications and consequences of this activity, both in terms of anthrax disease, and how this activity can be exploited to gain insight into other pathologic conditions.
anthrax; lethal factor; mitogen-activated protein kinase kinase; pathogenesis; metalloprotease; tumorigenesis; retinal neovascularization
Solid tumor growth is dependent on angiogenesis, the formation of neovasculature from existing vessels. Endothelial activation of the ERK1/2, JNK, and p38 mitogen activated protein kinase (MAPK) pathways is central to this process, and thus presents an attractive target for the development of angiogenesis inhibitors. Anthrax lethal toxin (LeTx) has potent catalytic MAPK inhibition activity. Preclinical studies showed LeTx induced potent tumor growth inhibition via the inhibition of xenograft vascularization. However, LeTx receptors and the essential furin-like activating proteases are expressed in many normal tissues, potentially limiting the specificity of LeTx as an anti-tumor agent. To circumvent nonspecific LeTx activation and simultaneously enhance tumor vascular targeting, a substrate preferably cleaved by the gelatinases class of matrix metalloproteinases (MMPs) was substituted for the furin LeTx activation site. In vivo efficacy studies demonstrated that this MMP-activated LeTx inhibited tumor xenografts growth via the reduced migration of endothelial cells into the tumor parenchyma. Here we have expanded on these initial findings by demonstrating that this MMP-activated LeTx reduces endothelial proangiogenic MMP expression, thus causing a diminished proteolytic capacity for extracellular matrix remodeling and endothelial differentiation into capillary networks. Additionally, our data suggests that inhibition of the JNK and p38, but not ERK1/2 pathways is significant in the anti-angiogenic activity of the MMP-activated LeTx. Collectively, these results support the clinical development of the MMP-activated LeTx for the treatment of solid tumors.
anthrax lethal toxin; lethal factor; matrix metalloproteinase; protective antigen; tumor angiogenesis; vascular endothelial growth factor
Anthrax Lethal Toxin (LeTx) demonstrates potent MAPK pathway inhibition and apoptosis in melanoma cells that harbor the activating V600E B-RAF mutation. LeTx is composed of two proteins, PA and LF. Uptake of the toxin into cells is dependent upon proteolytic activation of PA by the ubiquitously expressed furin or furin-like proteases. In order to circumvent nonspecific LeTx activation, a substrate preferably cleaved by gelatinases was substituted for the furin LeTx activation site. Here we have shown the toxicity of this MMP-activated LeTx is dependent on host cell surface MMP-2 and −9 activity as well as the presence of the activating V600E B-RAF mutation, making this toxin dual specific. This additional layer of tumor cell specificity would potentially decrease systemic toxicity from the reduction of nonspecific toxin activation while retaining anti-tumor efficacy in patients with V600E B-RAF melanomas. Moreover, our results indicate that cell surface-associated gelatinase expression can be used to predict sensitivity among V600E B-RAF melanomas. This finding will aid in the better selection of patients that will potentially respond to MMP-activated LeTx therapy.
anthrax lethal toxin; B-RAF; lethal factor; matrix metalloproteinase; protective antigen
Lethal factor, the enzymatic moiety of anthrax lethal toxin (LeTx) is a protease that inactivates mitogen activated protein kinase kinases (MEK or MKK). In vitro and in vivo studies demonstrate LeTx targets endothelial cells. However, the effects of LeTx on endothelial cells are incompletely characterized. To gain insight into this process we used a developmental model of vascularization in the murine retina. We hypothesized that application of LeTx would disrupt normal retinal vascularization, specifically during the angiogenic phase of vascular development. By immunoblotting and immunofluorescence microscopy we observed that MAPK activation occurs in a spatially and temporally regulated manner during retinal vascular development. Intravitreal administration of LeTx caused an early delay (4 d post injection) in retinal vascular development that was marked by reduced penetration of vessels into distal regions of the retina as well as failure of sprouting vessels to form the deep and intermediate plexuses within the inner retina. In contrast, later stages (8 d post injection) were characterized by the formation of abnormal vascular tufts that co-stained with phosphorylated MAPK in the outer retinal region. We also observed a significant increase in the levels of secreted VEGF in the vitreous 4 d and 8 d after LeTx injection. In contrast, the levels of over 50 cytokines other cytokines, including bFGF, EGF, MCP-1, and MMP-9, remained unchanged. Finally, co-injection of VEGF-neutralizing antibodies significantly decreased LeTx-induced neovascular growth. Our studies not only reveal that MAPK signaling plays a key role in retinal angiogenesis but also that perturbation of MAPK signaling by LeTx can profoundly alter vascular morphogenesis.
Anthrax toxin (AnTx) plays a key role in the pathogenesis of anthrax. AnTx is composed of three proteins: protective antigen (PA), edema factor, and lethal factor (LF). PA is not toxic but serves to bind cells and translocate the toxic edema factor or LF moieties to the cytosol. Recently, the low-density lipoprotein receptor–related protein LRP6 has been reported to mediate internalization and lethality of AnTx. Based on its similarity to LRP6, we hypothesized that LRP5 may also play a role in cellular uptake of AnTx. We assayed PA-dependent uptake of anthrax LF or a cytotoxic LF fusion protein (FP59) in cells and mice harboring targeted deletions of Lrp5 or Lrp6. Unexpectedly, we observed that uptake was unaltered in the presence or absence of either Lrp5 or Lrp6 expression. Moreover, we observed efficient PA-mediated uptake into anthrax toxin receptor (ANTXR)–deficient Chinese hamster ovary cells (PR230) that had been stably engineered to express either human ANTXR1 or human ANTXR2 in the presence or absence of siRNA specific for LRP5 or LRP6. Our results demonstrate that neither LRP5 nor LRP6 is necessary for PA-mediated internalization or lethality of anthrax lethal toxin.
The effects of many pathogenic bacteria are caused by the toxins they release. The toxin released by bacteria that cause anthrax is particularly fascinating since it is made of three different proteins: edema factor, lethal factor, and protective antigen (PA). On their own, each of these proteins is harmless, but when combined, they are deadly. This is because edema factor and lethal factor can exert their poisonous effects only after they have been moved into cells by PA. Determining exactly how PA does this is seen as a critical step in developing medicines that will fight anthrax. That is why a recent report suggesting that LRP6, an outer cell protein, was needed for PA to move the other toxin proteins into cells, was greeted with such interest. However, we now show that mice or cells lacking LRP6, or a related protein called LRP5, are still susceptible to anthrax toxin. The discovery that PA can move lethal factor and edema factor into cells without the help of LRP6 presents a significant challenge to the previously published model. These findings will help focus the efforts of scientists working on new ways to treat anthrax.