Enhanced metabolism as a common feature of cancer plasticity.

Cancer cells often rely on glycolytic metabolism in order to fulfill high demands of ATP and macromolecules for the sustained growth and proliferation. However, glycolysis is not necessarily the main source of energy for all cancer cells. Some of them rather depend on glutamine or lactate that favor the utilization of oxidative metabolic pathway. Different employment rate of metabolism creates variable products that participate in the formation of environmental milieu, which in turn triggers broad spectrum of cellular signaling pathways leading to migration, invasion, or proliferation. In this review we discuss different metabolic pathways promoted in tumor cells and describe the possibilities of their targeting as therapeutic strategies.

Ectodomain shedding of the hypoxia-induced carbonic anhydrase IX is a metalloprotease-dependent process regulated by TACE/ADAM17.

Carbonic anhydrase IX (CA IX) is a transmembrane protein whose expression is strongly induced by hypoxia in a broad spectrum of human tumours. It is a highly active enzyme functionally involved in both pH control and cell adhesion. Its presence in tumours usually indicates poor prognosis. Ectodomain of CA IX is detectable in the culture medium and body fluids of cancer patients, but the mechanism of its shedding has not been thoroughly investigated. Here, we analysed several cell lines with natural and ectopic expression of CA IX to show that its ectodomain release is sensitive to metalloprotease inhibitor batimastat (BB-94) and that hypoxia maintains the normal rate of basal shedding, thus leading to concomitant increase in cell-associated and extracellular CA IX levels. Using CHO-M2 cells defective in shedding, we demonstrated that the basal CA IX ectodomain release does not require a functional TNFalpha-converting enzyme (TACE/ADAM17), whereas the activation of CA IX shedding by both phorbol-12-myristate-13-acetate and pervanadate is TACE-dependent. Our results suggest that the cleavage of CA IX ectodomain is a regulated process that responds to physiological factors and signal transduction stimuli and may therefore contribute to adaptive changes in the protein composition of tumour cells and their microenvironment.

Characterization of the MN/CA 9 promoter proximal region: a role for specificity protein (SP) and activator protein 1 (AP1) factors.

MN/CA IX (MN) is a tumour-associated isoenzyme of the carbonic anhydrase family. Previous deletion analysis of the MN promoter established that protected regions (PRs) 1 and 2 are crucial for its transcriptional activity. Computer-assisted searching indicated putative binding sites for activator protein (AP) 2 and specificity protein (SP) 1 transcription factors, plus a CACCC box in PR1 and an AP1 site in PR2. PR1 produced four complexes in electrophoretic mobility-shift assay (EMSA) with HeLa nuclear extracts. Of these, three were completely competed with the SP1 and transforming growth factor-beta retinoblastoma control-element CACCC box (RCE) probes, whereas the AP2 probe competed against the same three complexes partially. Supershift EMSA identified SP1 in the complex 1 and SP3 in the complexes 2 and 4. Point mutations in the SP1 site abrogated the PR1 function, while mutations affecting the overlapping CACCC box/AP2 site in PR1 had minor effect on MN promoter activity. Block-replaced MN promoter mutants that had a consensus binding site (SP1 or AP2) or the RCE in place of PR1 demonstrated the stringent selectivity of the PR1 position as only the SP1 mutant reconstituted the MN promoter activity. The consensus SP1 probe generated the same SP1 and SP3 complexes as PR1 in EMSA; therefore we conclude that SP activity is both necessary and sufficient in the PR1 position. The critical role of AP1 in the PR2 position was confirmed by supershift of the PR2 complex with c-Fos antibody and markedly decreased activity of the construct with a mutated AP1 site. Detailed deletion analysis proved that PR1+PR2 account for 90% of the MN promoter activity, while neither PR1 nor PR2 on their own are sufficient for transactivation. Thus, synergistic co-operation between SP and AP1 factors bound to the adjacent PR1 and PR2, respectively, is necessary for MN transcriptional activity. The PR1+PR2 module also stimulated transcription from a heterologous promoter. The modulation of AP1 activity with PMA stimulated MN expression and activated the MN promoter, whereas inhibition of protein kinase C activity had no effect on MN expression in HeLa cells.