Abstract
Fhit protein is lost in cancers of most, perhaps all, cancer types; when restored, it can induce apoptosis and suppress tumorigenicity, as shown in vitro and in mouse tumor models in vivo. Following protein cross-linking and proteomics analyses, we characterized a Fhit protein complex involved in triggering Fhit-mediated apoptosis. The complex includes the heat-shock chaperonin pair, HSP60/10, which is likely involved in importing Fhit into the mitochondria, where it interacts with ferredoxin reductase, responsible for transferring electrons from NADPH to cytochrome P450 via ferredoxin, in electron transport chain complex III. Overexpression of Fhit protein in Fhit-deficient cancer cells modulates the production of intracellular reactive oxygen species, causing increased ROS, following peroxide treatment, with subsequent increased apoptosis of lung cancer cells under oxidative stress conditions; conversely, Fhit-negative cells escape ROS overproduction and ROS-induced apoptosis, likely carrying oxidative damage. Thus, characterization of Fhit-interacting proteins has identified direct effectors of a Fhit-mediated apoptotic signal pathway that is lost in many cancers. This is of translational interest considering the very recent emphasis in a number of high-profile publications, concerning the role of oxidative phosphorylation in the treatment of human cancers, and especially cancer stem cells that rely upon oxidative phosphorylation for survival. Additionally, we have shown that cells from a Fhit-deficient lung cancer cell line, are sensitive to killing by exposure to atovaquone, thought to act as a selective oxidative phosphorylation inhibitor by targeting the CoQ10 dependence of the mitochondrial complex III, while the Fhit-expressing sister clone is resistant to this treatment.
Highlights
The ~2-MB FHIT genomic locus straddles an active common fragile site at chromosome band 3p14.21,2
Purified proteins were treated with dithiothreitol (DTT) to cleave dithiobis(succinimidyl propionate) (DSP) and dissociate the complex, and digested by trypsin; protein constituents were identified by liquid-chromatography tandem mass spectrometry (LC-MS/MS) (Table 1 and Supplemental material) and six proteins were identified, all with mitochondrial localization: HSP60 and 10, ferredoxin reductase (Fdxr), malate dehydrogenase (Mdh), electron-transfer flavoprotein (Etfb), and mitochondrial aldehyde dehydrogenase 2 (Aldh2); HSP60 and HSP10 are distributed in the cytosol[25]
Since HSP60/10 complex acts as a chaperonin, we thought that interaction with this heat-shock protein complex might chaperone Fhit to the mitochondria, where Fdxr is important in electron transport
Summary
The ~2-MB FHIT genomic locus straddles an active common fragile site at chromosome band 3p14.21,2. Due to this genomic fragility, Fhit mRNA and/or protein expression is lost or reduced in large fractions of almost all types of human tumors, due to allelic loss, genomic rearrangement, promoter hypermethylation, or DNA damage checkpoint genes are already activated, in Official journal of the Cell Death Differentiation Association. Evidence of the loss of FHIT alleles occurs in normal-appearing bronchial epithelial cells of smokers, prior to pathologic changes or alterations in the expression of oncogenes or other suppressor genes[17,18,19]. To identify proteins that interact with Fhit to affect downstream apoptotic pathways, we cross-linked proteins within cells, after induced or viral-mediated Fhit overexpression in lung cancer cells, or endogenous expression in colon cancer cells, and characterized proteins associated with Fhit, and pathways affected by them
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