Abstract
We recently reported that Fhit is in a molecular complex with annexin A4 (ANXA4); following to their binding, Fhit delocalizes ANXA4 from plasma membrane to cytosol in paclitaxel-resistant lung cancer cells, thus restoring their chemosensitivity to the drug. Here, we demonstrate that Fhit physically interacts with A4 through its N-terminus; molecular dynamics simulations were performed on a 3D Fhit model to rationalize its mechanism of action. This approach allowed for the identification of the QHLIKPS heptapeptide (position 7 to 13 of the wild-type Fhit protein) as the smallest Fhit sequence still able to preserve its ability to bind ANXA4. Interestingly, Fhit peptide also recapitulates the property of the native protein in inhibiting Annexin A4 translocation from cytosol to plasma membrane in A549 and Calu-2 lung cancer cells treated with paclitaxel. Finally, the combination of Tat-Fhit peptide and paclitaxel synergistically increases the apoptotic rate of cultured lung cancer cells and blocks in vivo tumor formation.Our findings address to the identification of chemically simplified Fhit derivatives that mimic Fhit tumor suppressor functions; intriguingly, this approach might lead to the generation of novel anticancer drugs to be used in combination with conventional therapies in Fhit-negative tumors to prevent or delay chemoresistance.
Highlights
The FHIT gene encompasses the most common fragile site, FRA3B at 3p14.2 [1, 2] in human lymphoblasts; interestingly, this region is not the most fragile locus in epithelial cells [3], FHIT loss is commonly reported in human tumors of this derivation as an early event [4], through genetic inactivation [5] and through the silencing of its promoter due to hypermethylation [6].www.impactjournals.com/oncotargetSeveral evidences pointed at FHIT as a tumor suppressor gene; its genetic ablation in mice results both in an increase of spontaneous tumors and in a much higher susceptibility to develop carcinogeninduced tumors than wild-type counterparts [7]
As Fhit protein is virtually ubiquitously distributed into the cell [17, 18], later on we tried to isolate novel Fhit partners with oncogenic activity starting from cell membranes; we successfully identified annexin A4 (ANXA4) [19], a protein belonging to a superfamily of calcium-regulated and phospholipid membrane-binding proteins [20]
We recently begun to shed lights about Fhit protein partners unveiling novel pathways depending upon it; by using a proteomic approach we reported for the first time a Fhit complex including Hsp10, Hsp60, and ferredoxin reductase which underlies and supports the role of Fhit in apoptosis through the generation of free radicals in mitochondria [17]
Summary
The FHIT gene encompasses the most common fragile site, FRA3B at 3p14.2 [1, 2] in human lymphoblasts; interestingly, this region is not the most fragile locus in epithelial cells [3], FHIT loss is commonly reported in human tumors of this derivation as an early event [4], through genetic inactivation (i.e., deletions and translocations) [5] and through the silencing of its promoter due to hypermethylation [6].www.impactjournals.com/oncotargetSeveral evidences pointed at FHIT as a tumor suppressor gene; its genetic ablation in mice results both in an increase of spontaneous tumors and in a much higher susceptibility to develop carcinogeninduced tumors than wild-type counterparts [7]. FHIT restoration in FHIT-negative cancer cells of both epithelial origin and leukemias blocks in vivo tumor formation and triggers caspase-mediated apoptosis [10,11,12]. Hsp and Hsp take Fhit protein into the mitochondrion where it binds some mitochondrial proteins, including Fdxr; this protein complex induces the generation of reactive oxygen species (ROS) which, in turn, triggers apoptosis of Fhitnegative cancer cells [17]. Fhit restoration in Fhitnegative lung cancer cells blocks ANXA4 translocation from cytosol to the inner side of plasma membrane during paclitaxel administration, an effect that contribute to chemoresistance, sensitizing again cancer cells to the drug [19]
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