Mitochondrial activation by inhibition of PDKII suppresses HIF1a signaling and angiogenesis in cancer.

Most solid tumors are characterized by a metabolic shift from glucose oxidation to glycolysis, in part due to actively suppressed mitochondrial function, a state that favors resistance to apoptosis. Suppressed mitochondrial function may also contribute to the activation of hypoxia-inducible factor 1α (HIF1α) and angiogenesis. We have previously shown that the inhibitor of pyruvate dehydrogenase kinase (PDK) dichloroacetate (DCA) activates glucose oxidation and induces apoptosis in cancer cells in vitro and in vivo. We hypothesized that DCA will also reverse the 'pseudohypoxic' mitochondrial signals that lead to HIF1α activation in cancer, even in the absence of hypoxia and inhibit cancer angiogenesis. We show that inhibition of PDKII inhibits HIF1α in cancer cells using several techniques, including HIF1α luciferase reporter assays. Using pharmacologic and molecular approaches that suppress the prolyl-hydroxylase (PHD)-mediated inhibition of HIF1α, we show that DCA inhibits HIF1α by both a PHD-dependent mechanism (that involves a DCA-induced increase in the production of mitochondria-derived α-ketoglutarate) and a PHD-independent mechanism, involving activation of p53 via mitochondrial-derived H(2)O(2), as well as activation of GSK3ß. Effective inhibition of HIF1α is shown by a decrease in the expression of several HIF1α regulated gene products as well as inhibition of angiogenesis in vitro in matrigel assays. More importantly, in rat xenotransplant models of non-small cell lung cancer and breast cancer, we show effective inhibition of angiogenesis and tumor perfusion in vivo, assessed by contrast-enhanced ultrasonography, nuclear imaging techniques and histology. This work suggests that mitochondria-targeting metabolic modulators that increase pyruvate dehydrogenase activity, in addition to the recently described pro-apoptotic and anti-proliferative effects, suppress angiogenesis as well, normalizing the pseudo-hypoxic signals that lead to normoxic HIF1α activation in solid tumors.

Mutations in the S1 subunit of pertussis toxin that affect secretion.

Pertussis toxin is a member of the AB(5) family of toxins and is composed of five subunits (S1 to S5) present in a 1:1:1:2:1 ratio. Secretion is a complex process. Each subunit has a secretion signal that mediates transport to the periplasm, where processing and assembly occur. Secretion of the assembled 105-kDa toxin past the outer membrane is mediated by the nine proteins encoded in the ptl operon. Previous studies have shown that S1, the catalytically active A subunit of pertussis toxin, is necessary for efficient secretion, suggesting that a domain on S1 may be required for interaction with the secretion apparatus. Previously, recombinant S1 from four different mutants (serine 54 to glycine, serine 55 to glycine, serine 56 to glycine, and arginine 57 to lysine) was shown to retain catalytic activity. We introduced these mutations into Bordetella pertussis and monitored pertussis toxin production and secretion. No pertussis toxin was detected in the serine 54-to-glycine mutant. The other S1 mutants produced periplasmic pertussis toxin, but little pertussis toxin secretion was observed. The arginine 57-to-lysine mutant had the most dramatic secretion defect. It produced wild-type levels of periplasmic pertussis toxin but secreted only 8% as much toxin as the wild-type strain. This phenotype was similar to that observed for strains with mutations in the ptl genes, suggesting that this region may have a role in pertussis toxin secretion.

Identification of two bvg-repressed surface proteins of Bordetella pertussis.

Bordetella pertussis, the etiological agent of whooping cough, has the ability to modulate its phenotype in response to environmental conditions by using the BvgAS sensory transduction system which is encoded by the vir locus (now known as bvg). The BvgAS system is part of a large family of two-component sensory transduction systems which are common to a number of pathogenic bacteria. Although much is known about the proteins which exist in the B. pertussis virulent (X-mode or phase I) phenotype, relatively little is known about the proteins produced in the avirulent (C-mode or phase III) phenotype. We used sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing techniques to demonstrate the existence of at least 22 vir-repressed molecules which are increased in the avirulent phenotype. In addition, a series of monoclonal antibodies which are specific for the surface of avirulent B. pertussis were developed. Using immunological and protein techniques, we characterized two of these antigens as surface-exposed proteins. One of these antigens is expressed only in B. pertussis but not in the related species B. parapertussis and B. bronchiseptica. The other antigen is also present in B. parapertussis and B. bronchiseptica but is expressed at lower levels which are not regulated by bvg. The identification and characterization of vir-repressed proteins (and the genes which encode and regulate them) may help elucidate a physiological role for modulation of this obligate human pathogen.