Fhit Delocalizes Annexin A4 from Plasma Membrane to Cytosol and Sensitizes Lung Cancer Cells to Paclitaxel

<div><p>Fhit protein is lost or reduced in a large fraction of human tumors, and its restoration triggers apoptosis and suppresses tumor formation or progression in preclinical models. Here, we describe the identification of candidate Fhit-interacting proteins with cytosolic and plasma membrane localization. Among these, Annexin 4 (ANXA4) was validated by co-immunoprecipitation and confocal microscopy as a partner of this novel Fhit protein complex. Here we report that overexpression of Fhit prevents Annexin A4 translocation from cytosol to plasma membrane in A549 lung cancer cells treated with paclitaxel. Moreover, paclitaxel administration in combination with Ad<i>FHIT</i> acts synergistically to increase the apoptotic rate of tumor cells both <i>in vitro</i> and <i>in vivo</i> experiments.</p></div

Annexin 4 depletion, combined with Fhit overexpression and paclitaxel treatment induces inhibition of proliferation and triggers apoptosis.

<p>A. A549 cells were mock-transfected or transfected with Annexin 4 siRNAs (50 nM) or scrambled siRNAs (50 nM) for 72 h. Cells lysates were immunoblotted with Annexin 4 and Gapdh antibodies. B. A549 cells were mock transfected or transfected with Annexin 4 siRNAs (50 nM) or scrambled siRNA (50 nM), infected with Ad<i>FHIT</i> at MOI25 for 72 h, and then left untreated or treated with paclitaxel (800 nM). Cells were first counted at 12 h after paclitaxel treatment. Bar graphs show mean ± SEM for values from 3 experiments (* P<0,05). The Chou-Talalay methos was applied to calculate the nature of the combinations (CI<1, synergism). C. A549 cells were mock transfected or transfected with Annexin 4 siRNA (50 nM) or scrambled siRNAs (50 nM) for 72 h, then untreated or treated with paclitaxel. Cells were first counted 12 h after paclitaxel treatment. Bar graphs show mean ± SEM for values from 3 experiments (* P<0,05). The Chou-Talalay methos was applied to calculate the nature of the combinations (CI<1, synergism). D. A549 cells, treated as described in B and C, were analyzed by TUNEL assay.</p

Fhit overexpression blocks Annexin 4 translocation from cytosol to plasma membrane.

<p><b>A-C</b>. A549 and Calu-2 lung cancer cells were infected with Ad<i>GFP</i> or Ad<i>FHIT</i> at MOI50; 48 h later, cells were mock-treated or treated with paclitaxel (800 nM) for an additional 24 h. Proteins from cytosolic and membrane fractions were separated on a polyacrylamide gel, transferred to nitrocellulose filter, and probed with Annexin 4 antibody. Gapdh and E-cadherin were used to normalize protein loading of cytosolic and plasma membrane proteins, respectively. <b>B-D</b>. A549 and Calu-2 lung cancer cells were infected with Ad<i>GFP</i> or Ad<i>FHIT</i>-wild-type; 48 h later, cells were treated with paclitaxel (800 nM) for an additional 24 h. Proteins from cytosolic and membrane fractions were separated on a polyacrylamide gel, transferred to nitrocellulose filter, and probed with Annexin 1 and MDR (multi-drug resistance protein) antibodies. Gapdh and E-cadherin were used to normalize protein loading of cytosolic and plasma membrane proteins, respectively.</p

Fhit/Annexin 4 interaction plus paclitaxel induced tumor regression in a preclinical model of lung cancer.

<p><b>A</b>. Nude mice were subcutaneously injected with 1×10⁷ A549 cells. Some groups (n = 5 mice/group) were injected with mock treated cells, others with cells transfected with Annexin 4 siRNA or infected with Ad<i>FHIT</i> (MOI5 or MOI50) or combinations of both. When tumors reached 15 mm diameter, mice were mock-treated, treated with DMSO or treated with a single IV administration of 40 mg/kg paclitaxel; mice were monitored on a regular basis. Three days after PTX, mice were sacrificed and tumors were evaluated by weight. Bar graphs show mean ± SEM for values from 5 mice (* P<0,05). The Chou-Talalay methos was applied to calculate the nature of the combinations (CI<1, synergism). <b>B</b>. Tumor volumes are reported over time.</p

List of putative interactors of Fhit protein identified by mass spectrometry.

<p>List of putative interactors of Fhit protein identified by mass spectrometry.</p

Fhit physically interacts with Annexin 4.

<p><b>A</b>. A549 lung cancer cells were infected with Ad<i>FHIT</i>-wild type or Ad<i>FHIT-</i>His6 at MOI50; 48 h after infection, cells were treated with DSP and lysed with Mem-PER Eukaryotic Membrane Protein Extraction Kit to provide total lysates enriched in membrane fraction. Total lysates were immunoprecipitated with nickel beads. Immunoprecipitates were analyzed by immunoblotting (IB) with anti-Fhit and anti-Annexin A4 antibodies. <b>B</b>. A549 cells were transiently transfected with an expression plasmid encoding mammalian <i>Annexin4-</i>V5 (8 µg). 48 h after transfection, cells were treated with DSP and total lysates were immunoprecipitated (IP) with anti-V5 antibody. The immunoprecipitates were probed by immunoblotting (IB) with anti-Fhit and anti-Annexin 4 antibodies. <b>C-D</b>. HEK293 cells were mock tretated or tretaed with paclitaxel for 24 hrs (800 nM) and lysed. Total protein lysates were Immunoprecipited with IgG, Fhit and Annexin 4 antibodies, as indicated. The detection of endogenous Annexin 4 and Fhit was performed without the use of the DSP cross-linker.</p

Fhit overexpression and paclitaxel treatment induces synergistic proliferation inhibition.

<p><b>A-B</b>. A549 and Calu-2 cells were mock-infected, Ad<i>GFP</i> or Ad<i>FHIT</i> infected cells at MOI10 for 24 h, then treated with or without 800 nM paclitaxel for additional 6 h and counted. Bar graphs show mean ± SEM for values from three different experiments (* P<0,05). The Chou-Talalay methos was applied to calculate the nature of the combinations (CI<1, synergism). <b>C</b>. A549 cells were either mock-infected or infected with Ad<i>GFP</i> or Ad<i>FHIT</i>, or left untreated or treated with 800 nM paclitaxel, and then analyzed by flow cytometry.</p