Fhit Interaction with Ferredoxin Reductase Triggers Generation of Reactive Oxygen Species and Apoptosis of Cancer Cells

Francesco Trapasso,Flavia Pichiorri,Marco Gaspari,Tiziana Palumbo,Rami I Aqeilan,Eugenio Gaudio,Hiroshi Okumura,Rodolfo Iuliano,Giampiero Di Leva,Muller Fabbri,David E Birk,Cinzia Raso,Kari Green-Church,Luigi G Spagnoli,Salvatore Venuta,Kay Huebner,Carlo M Croce

doi:10.1074/jbc.m709062200

Abstract

Fhit protein is lost in most cancers, its restoration suppresses tumorigenicity, and virus-mediated FHIT gene therapy induces apoptosis and suppresses tumors in preclinical models. We have used protein cross-linking and proteomics methods to characterize a Fhit protein complex involved in triggering Fhit-mediated apoptosis. The complex includes Hsp60 and Hsp10 that mediate Fhit stability and may affect import into mitochondria, where it interacts with ferredoxin reductase, responsible for transferring electrons from NADPH to cytochrome P450 via ferredoxin. Viral-mediated Fhit restoration increases production of intracellular reactive oxygen species, followed by increased apoptosis of lung cancer cells under oxidative stress conditions; conversely, Fhit-negative cells escape apoptosis, carrying serious oxidative DNA damage that may contribute to an increased mutation rate. Characterization of Fhit interacting proteins has identified direct effectors of the Fhit-mediated apoptotic pathway that is lost in most cancers through loss of Fhit.

Highlights

Fhit overexpression in lung cancer cells, and characterized proThe FHIT gene encompasses the most active common fragile teins associated with Fhit and the pathways affected by them. site at chromosome 3p14.2 [1, 2]
ferredoxin reductase (Fdxr), were identified; subcellular location of these proteins suggested that mitochondria might be foci of
Hsp60/Hsp10 after AdFHIT infection suggests that the Hsp complex may be important for Fhit stability, and possibly for its correct folding to import it into mitochondria, prior to activation of the apoptotic pathway, a suggestion we investigated by knocking down expression of Hsp60, Hsp10, or both in AdFHIT-infected lung cancer cells; Fhit stability was assessed after CHX chase in H1299 D1 cells, the lung cancer cell line expressing inducible Fhit

Summary

Introduction

Fhit overexpression in lung cancer cells, and characterized proThe FHIT gene encompasses the most active common fragile teins associated with Fhit and the pathways affected by them. site at chromosome 3p14.2 [1, 2]. Fhit overexpression in lung cancer cells, and characterized proThe FHIT gene encompasses the most active common fragile teins associated with Fhit and the pathways affected by them. Site at chromosome 3p14.2 [1, 2]. Fhit expression is lost or reduced in a large fraction of most types of human tumors due to allelic loss, genomic rearrangement, promoter hypermethylation, or combinations thereof [3, 4]. Fhit knock-out mice show increased susceptibility to cancer development [5, 6] and FHIT gene therapy prevents tumors in carcinogen-exposed Fhit-deficient mice [7, 8]. Fhit restoration by stable transfection in cancer cells has little effect in vitro, unless cells are exposed to

Methods

Results

Conclusion