Since the very first moment of its discovery in 1996 [1], the FHIT (Fragile Histidine Triad) gene product appeared as a very intriguing molecule to be investigated in the pathogenesis of cancer. In fact, FHIT encompasses the most common fragile site in human [2], whose genetic alterations, leading to the loss of FHIT expression, have been reported in the majority of human cancers [3]. Other than genetic lesions, FHIT expression in both solid and hematopoietic malignancies is also impaired by its promoter hypermethylation [4, 5], making Fhit protein virtually completely lost in tumors. Moreover, several reports have intriguingly pointed to Fhit loss as a very early event in epithelial tumorigenesis (presumably, the earliest event in smoking-related lung cancer) [3]. These findings, along with both chemically induced and spontaneous predisposition to cancer development of Fhit+/- and Fhit-/- mice, respectively [6, 7], have encouraged the application of a gene therapy approach in several experimental models of cancer, both in vitro and in vivo. We successfully demonstrated that Fhit restoration in cancer cells through recombinant adeno- or adenoassociate viruses was able to trigger apoptosis in a number of cancer types, including esophageal, pancreatic, mammary cancer, and even leukemia, and to block their in vivo tumor formation [8-11]. Moreover, Fhit protein was not only curative in Fhit+/- mice bearing NMBA-induced forestomach tumors [12] but it could also prevent tumor formation in Fhit+/- mice treated with the same carcinogen [13, 14]. These results were very interesting as they represented the proof-of- principle that FHIT was a therapeutic gene indeed. Fhit protein function is still partly a mystery; for long time we only knew that it was an enzyme belonging to the HIT (Histidine Triad) protein family, a class of molecules involved in the hydrolysis of dinucleoside three- and tetraphosphate [3]. In order to investigate the role of Fhit hydrolase activity in tumor suppression, we planned an apoptosis quantitative assay based on the design of FHIT alleles driven by recombinant adenoviruses. We proved that the Fhit mutants able to bind the substrate but with impaired catalytic activity could still efficiently trigger apoptosis of cancer cells; also, apoptosis was still observed with Fhit mutants unable to bind the substrate, even though to a much lower extent compared to the wild-type protein [15, 16], Letter to the Editors thus suggesting that Fhit tumor suppression activity in cancer cells could presumably be dependent by different and independent molecular pathways. However, because of the failure to identify protein partners through conventional approaches, no Fhit pathways have ever been described for more than a decade after its discovery. Therefore, this limitation has contributed to lower the interest toward a molecule whose importance in human and experimental tumorigenesis was clearly established. Finally, in 2008 we published a pivotal study demonstrating for the first time the existence of a Fhit protein complex [17]. We designed an elegant proteomic-based approach in order to identify the Fhit- interacting molecules once Fhit-mediated apoptosis was triggered in cancer cells after adenovirus-mediated FHIT restoration. Briefly, a tagged Fhit protein was expressed in lung cancer cells through a recombinant adenovirus; the candidate Fhit protein complex was then stabilized by using DSP, a vital photo-crosslinker able to generate disulfide bonds among the lysines of interacting proteins. Total cell extracts were then purified, digested and used to run a mass-spectrometry analysis. The list of candidate proteins was composed of six proteins only; all of them were tracked in mitochondria, including Hsp10 and Hsp60, also detected in the cytosol. These results described for the first time Fhit mitochondrial subcellular localization through the shuttling of Hsp60 and Hsp10 and, surprisingly, directly put Fhit protein in a pathway involving molecules, such ad ferredoxin reductase (Fdxr), important in the generation of reactive oxygen species (ROS) as a result of the activity of the respiratory chain. This aspect is very important in the physiology of a normal cell, as free radicals represent a protective mechanism toward cell injuries of different nature; in fact, their increase observed during exposure to noxious agents can address normal somatic cells to apoptosis, thus avoiding DNA alterations potentially leading to cancer [18]. These considerations put FHIT in a crucial position in an initiated cell, as its loss in the preneoplastic genome predisposes to reduced apoptosis; this allows, in turn, for the accumulation of further genetic lesions because of the reduction in the efficiency of the intrinsic pathway of apoptosis, plausibly due to the reduced extent of free radicals The Fhit protein: an opportunity to overcome chemoresistance Eugenio Gaudio, Francesco Paduano, Carlo M. Croce, Francesco Trapasso www.aging-us.com AGING 2016, Vol. 8, No. 10 www.aging‐us.com 1 AGING (Albany NY)