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Efficient targeting of head and neck squamous cell carcinoma by systemic administration of a dual uPA and MMP-activated engineered anthrax toxin.

Schafer JM, Peters DE, Morley T, Liu S, Molinolo AA, Leppla SH, Bugge TH - PLoS ONE (2011)

Bottom Line: HNSCC is characterized by the upregulation of a large number of proteolytic enzymes, including urokinase plasminogen activator (uPA) and an assortment of matrix metalloproteinases (MMPs) that may be expressed by tumor cells, by tumor-supporting stromal cells or by both.We found that this toxin displayed strong systemic anti-tumor activity towards a variety of xenografted human HNSCC cell lines by inducing apoptotic and necrotic tumor cell death, and by impairing tumor cell proliferation and angiogenesis.This intercomplementing toxin warrants further investigation as an anti-HNSCC agent.

View Article: PubMed Central - PubMed

Affiliation: Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Although considerable progress has been made in elucidating the etiology of the disease, the prognosis for individuals diagnosed with HNSCC remains poor, underscoring the need for development of additional treatment modalities. HNSCC is characterized by the upregulation of a large number of proteolytic enzymes, including urokinase plasminogen activator (uPA) and an assortment of matrix metalloproteinases (MMPs) that may be expressed by tumor cells, by tumor-supporting stromal cells or by both. Here we explored the use of an intercomplementing anthrax toxin that requires combined cell surface uPA and MMP activities for cellular intoxication and specifically targets the ERK/MAPK pathway for the treatment of HNSCC. We found that this toxin displayed strong systemic anti-tumor activity towards a variety of xenografted human HNSCC cell lines by inducing apoptotic and necrotic tumor cell death, and by impairing tumor cell proliferation and angiogenesis. Interestingly, the human HNSCC cell lines were insensitive to the intercomplementing toxin when cultured ex vivo, suggesting that either the toxin targets the tumor-supporting stromal cell compartment or that the tumor cell requirement for ERK/MAPK signaling differs in vivo and ex vivo. This intercomplementing toxin warrants further investigation as an anti-HNSCC agent.

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Cytotoxicity of intercomplementing anthrax lethal toxins to human HNSCC cell lines.Cal27 (blue triangles), Hep2 (purple triangles), HN6 (green squares), HN12 (purple squares), and HN30 (yellow diamonds) cells were incubated with increasing concentrations of wildtype PA in combination with FP59 (A), intercomplementing PA (PA-U2-R200A + PA-L1-I210A) in the presence of FP59 (B), wildtype PA with LF (C) or intercomplementing PA with LF (D) for 48 h. The cell viability was then measured using an MTT assay. HT29 colon carcinoma (grey circles) and HeLa (red circles) cells were used as a positive and negative controls, respectively [52], [74]. Cell survival is expressed as mean viability ± standard deviation of the mean.
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pone-0020532-g001: Cytotoxicity of intercomplementing anthrax lethal toxins to human HNSCC cell lines.Cal27 (blue triangles), Hep2 (purple triangles), HN6 (green squares), HN12 (purple squares), and HN30 (yellow diamonds) cells were incubated with increasing concentrations of wildtype PA in combination with FP59 (A), intercomplementing PA (PA-U2-R200A + PA-L1-I210A) in the presence of FP59 (B), wildtype PA with LF (C) or intercomplementing PA with LF (D) for 48 h. The cell viability was then measured using an MTT assay. HT29 colon carcinoma (grey circles) and HeLa (red circles) cells were used as a positive and negative controls, respectively [52], [74]. Cell survival is expressed as mean viability ± standard deviation of the mean.

Mentions: We first explored if HNSCC cells express functional anthrax toxin receptors by exposing the five human HNSCC cell lines, Cal27, Hep2, HN6, HN12, and HN30 to increasing concentrations of PA in combination with 1.9 nM FP59 (Figure 1A). FP59 is a fusion protein consisting of LF residues 1–254 with the ADP-ribosylation domain of Pseudomonas exotoxin A. When translocated to the cytoplasm via PA, FP59 efficiently kills all cells by ADP-ribosylation and inhibition of translation elongation factor 2 [64]. PA in combination with FP59 killed all HNSCC cell lines with an LD50 ranging from less than 7 to 400 pM demonstrating the presence of functional anthrax toxin receptors. To assess if these HNSCC cell lines also express both functional uPA and MMP cell surface proteolytic activity, we next exposed the five HNSCC cell lines to the same concentration of FP59 in combination with increasing concentrations of intercomplementing PA (PA-U2-R200A + PA-L1-I210A) (Figure 1B). Three of the HNSCC cell lines (Cal27, HN6, HN12) were sensitive to the intercomplementing toxin (LD50, 0.5 nM to 8 nM), demonstrating functional uPA and MMP expression, while the two other cell lines, Hep2 and HN30 were resistant, indicating the absence of either functional uPA or MMP activity, or both. We next determined if the five HNSCC cell lines were dependent on a functional MEK/MAPK pathway for growth in culture by incubating them with 5.5 nM LF in combination with increasing concentrations of either wildtype PA (Figure 1C) or intercomplementing PA (Figure 1D). None of the HNSCC cell lines were sensitive to the two toxin combinations, showing that MEK activity is dispensable for HNSCC cell growth in culture.


Efficient targeting of head and neck squamous cell carcinoma by systemic administration of a dual uPA and MMP-activated engineered anthrax toxin.

Schafer JM, Peters DE, Morley T, Liu S, Molinolo AA, Leppla SH, Bugge TH - PLoS ONE (2011)

Cytotoxicity of intercomplementing anthrax lethal toxins to human HNSCC cell lines.Cal27 (blue triangles), Hep2 (purple triangles), HN6 (green squares), HN12 (purple squares), and HN30 (yellow diamonds) cells were incubated with increasing concentrations of wildtype PA in combination with FP59 (A), intercomplementing PA (PA-U2-R200A + PA-L1-I210A) in the presence of FP59 (B), wildtype PA with LF (C) or intercomplementing PA with LF (D) for 48 h. The cell viability was then measured using an MTT assay. HT29 colon carcinoma (grey circles) and HeLa (red circles) cells were used as a positive and negative controls, respectively [52], [74]. Cell survival is expressed as mean viability ± standard deviation of the mean.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105081&req=5

pone-0020532-g001: Cytotoxicity of intercomplementing anthrax lethal toxins to human HNSCC cell lines.Cal27 (blue triangles), Hep2 (purple triangles), HN6 (green squares), HN12 (purple squares), and HN30 (yellow diamonds) cells were incubated with increasing concentrations of wildtype PA in combination with FP59 (A), intercomplementing PA (PA-U2-R200A + PA-L1-I210A) in the presence of FP59 (B), wildtype PA with LF (C) or intercomplementing PA with LF (D) for 48 h. The cell viability was then measured using an MTT assay. HT29 colon carcinoma (grey circles) and HeLa (red circles) cells were used as a positive and negative controls, respectively [52], [74]. Cell survival is expressed as mean viability ± standard deviation of the mean.
Mentions: We first explored if HNSCC cells express functional anthrax toxin receptors by exposing the five human HNSCC cell lines, Cal27, Hep2, HN6, HN12, and HN30 to increasing concentrations of PA in combination with 1.9 nM FP59 (Figure 1A). FP59 is a fusion protein consisting of LF residues 1–254 with the ADP-ribosylation domain of Pseudomonas exotoxin A. When translocated to the cytoplasm via PA, FP59 efficiently kills all cells by ADP-ribosylation and inhibition of translation elongation factor 2 [64]. PA in combination with FP59 killed all HNSCC cell lines with an LD50 ranging from less than 7 to 400 pM demonstrating the presence of functional anthrax toxin receptors. To assess if these HNSCC cell lines also express both functional uPA and MMP cell surface proteolytic activity, we next exposed the five HNSCC cell lines to the same concentration of FP59 in combination with increasing concentrations of intercomplementing PA (PA-U2-R200A + PA-L1-I210A) (Figure 1B). Three of the HNSCC cell lines (Cal27, HN6, HN12) were sensitive to the intercomplementing toxin (LD50, 0.5 nM to 8 nM), demonstrating functional uPA and MMP expression, while the two other cell lines, Hep2 and HN30 were resistant, indicating the absence of either functional uPA or MMP activity, or both. We next determined if the five HNSCC cell lines were dependent on a functional MEK/MAPK pathway for growth in culture by incubating them with 5.5 nM LF in combination with increasing concentrations of either wildtype PA (Figure 1C) or intercomplementing PA (Figure 1D). None of the HNSCC cell lines were sensitive to the two toxin combinations, showing that MEK activity is dispensable for HNSCC cell growth in culture.

Bottom Line: HNSCC is characterized by the upregulation of a large number of proteolytic enzymes, including urokinase plasminogen activator (uPA) and an assortment of matrix metalloproteinases (MMPs) that may be expressed by tumor cells, by tumor-supporting stromal cells or by both.We found that this toxin displayed strong systemic anti-tumor activity towards a variety of xenografted human HNSCC cell lines by inducing apoptotic and necrotic tumor cell death, and by impairing tumor cell proliferation and angiogenesis.This intercomplementing toxin warrants further investigation as an anti-HNSCC agent.

View Article: PubMed Central - PubMed

Affiliation: Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Although considerable progress has been made in elucidating the etiology of the disease, the prognosis for individuals diagnosed with HNSCC remains poor, underscoring the need for development of additional treatment modalities. HNSCC is characterized by the upregulation of a large number of proteolytic enzymes, including urokinase plasminogen activator (uPA) and an assortment of matrix metalloproteinases (MMPs) that may be expressed by tumor cells, by tumor-supporting stromal cells or by both. Here we explored the use of an intercomplementing anthrax toxin that requires combined cell surface uPA and MMP activities for cellular intoxication and specifically targets the ERK/MAPK pathway for the treatment of HNSCC. We found that this toxin displayed strong systemic anti-tumor activity towards a variety of xenografted human HNSCC cell lines by inducing apoptotic and necrotic tumor cell death, and by impairing tumor cell proliferation and angiogenesis. Interestingly, the human HNSCC cell lines were insensitive to the intercomplementing toxin when cultured ex vivo, suggesting that either the toxin targets the tumor-supporting stromal cell compartment or that the tumor cell requirement for ERK/MAPK signaling differs in vivo and ex vivo. This intercomplementing toxin warrants further investigation as an anti-HNSCC agent.

Show MeSH
Related in: MedlinePlus