Nicholas S. Duesbery

Apoptosis and melanogenesis in human melanoma cells induced by anthrax lethal factor inactivation of mitogen-activated protein kinase kinase

Lethal factor, the principal virulence factor of Bacillus anthracis, inhibits mitogen-activated protein kinase (MAPK) signaling by proteolytically cleaving MAPK kinases. Edema factor, another component of anthrax toxin, is an adenylate cyclase, which increases intracellular cAMP. Inhibition of MAPK signaling with either anthrax lethal toxin (LeTx) or small molecule MAPK kinase inhibitors triggers apoptosis in human melanoma cells. Normal melanocytes do not undergo apoptosis in response to MAPK inhibition but arrest in the G1 phase of the cell cycle. Importantly, in vivo treatment of human melanoma xenograft tumors in athymic nude mice with LeTx results in significant or complete tumor regression without apparent side effects, suggesting that inhibiting the MAPK signaling pathway may be a useful strategy for treating melanoma. Additionally, interrupting MAPK signaling with LeTx and elevating cAMP with anthrax edema toxin in both melanoma cells and melanocytes lead to dramatic melanin production, perhaps explaining the formation of blackened eschars in cutaneous anthrax.

Inhibition of MAPK kinase signaling pathways suppressed renal cell carcinoma growth and angiogenesis in vivo.

The mitogen-activated protein kinase (MAPK) signaling pathways play essential roles in cell proliferation and differentiation. Recent studies also show the activation of MAPK signaling pathways in tumorigenesis, metastasis, and angiogenesis of multiple human malignancies, including renal cell carcinoma (RCC). To assess the role of this pathway in regulating the proliferation and survival of RCC cells, we first examined the expression of MAPK kinase (MKK) and MAPK in clear cell RCC and confirmed the overexpression of MKK1 and extracellular signal-regulated kinase 2 (ERK2) in these tumors. We then tested the effects of pharmacologic inhibition of MKK on human RCC cell lines, both in vitro and in vivo, using anthrax lethal toxin (LeTx), which cleaves and inactivates several MKKs. Western blotting showed that the phosphorylation levels of ERK, c-Jun-NH(2) kinase, and p38 MAPK decreased after 72 h of LeTx treatment. Exposure to LeTx for 72 h reduced cell proliferation by 20% without significant effects on cell cycle distribution and apoptosis. Anchorage-independent growth of RCC cells was dramatically inhibited by LeTx. In vivo studies showed that tumor growth of RCC xenografts could be suppressed by LeTx. Extensive necrosis and decreased tumor neovascularization were observed after LeTx treatment. LeTx also showed direct inhibition of proliferation of endothelial cells in vitro. Our results suggest that suppression of one or more MAPK signaling pathways may inhibit RCC growth through the disruption of tumor vasculature.

Sensitizing Anthrax Lethal Toxin-resistant Macrophages to Lethal Toxin-induced Killing by Tumor Necrosis Factor-α

Macrophages from different inbred mouse strains exhibit striking differences in their sensitivity to anthrax lethal toxin (LeTx)-induced cytolysis. Although LeTx-induced cytolysis of macrophages plays an important role in the outcome of anthrax infection, the sensitivity of macrophages in vitrodoes not correlate with in vivo susceptibility to infection of Bacillus anthracis. This divergence suggests that additional factors other than LeTx are involved in the cytolysis of LeTx-resistant macrophages in vivo. We found that LeTx-resistant macrophages became sensitive to LeTx-induced cytolysis when these cells were activated by bacterial components. Tumor necrosis factor-α induced by bacterial components was a key factor that cooperated with LeTx in inducing LeTx-resistant macrophage death. Tumor necrosis factor-α/LeTx-induced death of LeTx-resistant macrophages was dependent on mTor (mammalian targetof rapamycin), but independent of caspases. Our data indicate that host responses to anthrax infection contribute to cytolysis of LeTx- resistant macrophages.

BRAF status and mitogen-activated protein/extracellular signal-regulated kinase kinase 1/2 activity indicate sensitivity of melanoma cells to anthrax lethal toxin

Anthrax lethal toxin, composed of protective antigen and lethal factor, was tested for cytotoxicity to human melanoma cell lines and normal human cells. Eleven of 18 melanoma cell lines were sensitive to anthrax lethal toxin (IC50 < 400 pmol/L) and 10 of these 11 sensitive cell lines carried the V599E BRAF mutation. Most normal cell types (10 of 15) were not sensitive to anthrax lethal toxin and only 5 of 15 normal human cell types were sensitive to anthrax lethal toxin (IC50 < 400 pmol/L). These cells included monocytes and a subset of endothelial cells. In both melanoma cell lines and normal cells, anthrax toxin receptor expression levels did not correlate with anthrax lethal toxin cytotoxicity. Furthermore, an anthrax toxin receptor–deficient cell line (PR230) did not show any enhanced sensitivity to anthrax lethal toxin when transfected with anthrax toxin receptor. Anthrax lethal toxin toxicity correlated with elevated phosphorylation levels of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) 1/2 in both melanoma cell lines and normal cells. Anthrax lethal toxin–sensitive melanoma cell lines and normal cells had higher phospho-MEK1/2 levels than anthrax lethal toxin–resistant melanoma cell lines and normal tissue types. U0126, a specific MEK1/2 inhibitor, was not toxic to anthrax lethal toxin–resistant melanoma cell lines but was toxic to 8 of 11 anthrax lethal toxin–sensitive cell lines. These results show that anthrax lethal toxin toxicity correlates with elevated levels of active MEK1/2 pathway but not with anthrax toxin receptor expression levels in both normal and malignant tissues. Anthrax lethal toxin may be a useful therapeutic for melanoma patients, especially those carrying the V599E BRAF mutation with constitutive activation of the mitogen-activated protein kinase pathway.

LRP5 and LRP6 Are Not Required for Protective Antigen-Mediated Internalization or Lethality of Anthrax Lethal Toxin

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.

Xp38γ/SAPK3 promotes meiotic G2/M transition in Xenopus oocytes and activates Cdc25C

We have studied the role of p38 mitogen‐activated protein kinases (MAPKs) in the meiotic maturation of Xenopus oocytes. Overexpression of a constitutively active mutant of the p38 activator MKK6 accelerates progesterone‐induced maturation. Immunoprecipit ation experiments indicate that p38γ/SAPK3 is the major p38 activated by MKK6 in the oocytes. We have cloned Xenopus p38γ (Xp38γ) and show that co‐expression of active MKK6 with Xp38γ induces oocyte maturation in the absence of progesterone. The maturation induced by Xp38γ requires neither protein synthesis nor activation of the p42 MAPK–p90Rsk pathway, but it is blocked by cAMP‐dependent protein kinase. A role for the endogenous Xp38γ in progesterone‐induced maturation is supported by the inhibitory effect of kinase‐dead mutants of MKK6 and Xp38γ. Furthermore, MKK6 can rescue the inhibition of oocyte maturation by anthrax lethal factor, a protease that inactivates MAPK kinases. We also show that Xp38γ can activate the phosphatase XCdc25C, and we identified Ser205 of XCdc25C as a major phosphorylation site for Xp38γ. Our results indicate that phosphorylation of XCdc25C by Xp38γ/SAPK3 is important for the meiotic G2/M progression of Xenopus oocytes.

MEK Wars, a new front in the battle against cancer

A specific inhibitor of the mitogen-activated protein kinase (MAPK or ERK) pathway is introduced as a new member in the growing search for cytostatic drugs that block tumor growth (pages 810–816).

Pathophysiology of anthrax.

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.

MEK genomics in development and disease

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.

Mitogen-activated protein kinase kinase signaling promotes growth and vascularization of fibrosarcoma.

We hypothesized that signaling through multiple mitogen-activated protein kinase (MAPK) kinase (MKK) pathways is essential for the growth and vascularization of soft-tissue sarcomas, which are malignant tumors derived from mesenchymal tissues. We tested this using HT-1080, NCI, and Shac fibrosarcoma-derived cell lines and anthrax lethal toxin (LeTx), a bacterial toxin that inactivates MKKs. Western blots confirmed that LeTx treatment reduced the levels of phosphorylated extracellular signal-regulated kinase and p38 MAPK in vitro. Although short treatments with LeTx only modestly affected cell proliferation, sustained treatment markedly reduced cell numbers. LeTx also substantially inhibited the extracellular release of angioproliferative factors including vascular endothelial growth factor, interleukin-8, and basic fibroblast growth factor. Similar results were obtained with cell lines derived from malignant fibrous histiocytomas, leiomyosarcomas, and liposarcomas. In vivo, LeTx decreased MAPK activity and blocked fibrosarcoma growth. Growth inhibition correlated with decreased cellular proliferation and extensive necrosis, and it was accompanied by a decrease in tumor mean vessel density as well as a reduction in serum expression of angioproliferative cytokines. Vital imaging using high-resolution ultrasound enhanced with contrast microbubbles revealed that the effects of LeTx on tumor perfusion were remarkably rapid (<24 h) and resulted in a marked reduction of perfusion within the tumor but not in nontumor tissues. These results are consistent with our initial hypothesis and lead us to propose that MKK inhibition by LeTx is a broadly effective strategy for targeting neovascularization in fibrosarcomas and other similar proliferative lesions. [Mol Cancer Ther 2008;7(3):648–58]