miR-194 targets the talin2 gene and downregulates talin2 protein levels.

<p>(<b>A</b>) Alignment of miR-194 with <i>talin2</i> (<i>TLN2</i>) 3′-UTRs. Complementary sequences of miR-194 and mammalian <i>talin2</i> 3′-UTRs are marked in Bold. The seed sequences of miR-194 are underlined. Has, human; Ptr, pan troglotydes; Mmu, mus musculus; Rno, rat; Ocu, rabbit; Eeu, hedgehog; Cfa, dog; Fca, cat; Eca, horse; Laf, elephant. The underlined seed nucleotides were deleted in the <i>talin2</i> 3′-UTR mutant reporter construct described in (B). (<b>B</b>) Effect of miR-194 expression on the luciferase activities of wild-type and mutated talin2 3′-UTR reporters. MDA-MB-361 cells were transiently transfected with a miR Control or miR-194 precursor for 36 hrs. Luciferase activity was determined using a dual luciferase assay. ** <i>p</i><0.01. (<b>C</b>) Effect of miR-194 expression on talin2 protein levels in SKBr3 cells. SKBr3 cells were transiently transfected with a miR-194 precursor or a control miRNA (miR CTRL) for 48 hrs. Total protein was prepared and subjected to Western blotting. (<b>D</b>) Effect of talin2 downregulation on cell migration in SKBr3 cells. SKBr3 cells were transiently transfected with two siTalin2 (siTLN2 #7 and #8) or its control siRNA (siCTRL) for 48 hrs and then motility was measured overnight in a Transwell assay. <b>*</b> p<0.05 compared to siCTRL. (<b>E</b>) Effect of talin2 downregulation on cell invasion. SKBr3 cells were transiently transfected with two siTalin2 (siTLN2 #7 and #8) or its control siRNA (siCTRL) for 48 hrs and then invasion assay was performed. <b>*</b> p<0.05 compared to siCTRL. (<b>F</b>) Validation of talin2 siRNA efficacy. SKBr3 cells were transiently transfected with two siTalin2 (siTLN2 #7 and #8) or its control siRNA (siCTRL) or the transfection reagent only (mock) for 48 hrs and total protein was prepared. Western blotting was performed with a talin2 antibody.</p

Trastuzumab treatment downregulates talin2 protein expression and inhibits breast cancer cell migration.

<p>(<b>A</b>) Effect of trastuzumab (Tras) on cell migration in a scratch assay. BT474 and SKBr3 cells were seeded in 6-well plates. After cells had grown to confluence, a scratch was made in the monolayer. Cells were treated with trastuzumab (Tras) or control hIgG (10 µg/ml) for 72 hrs. Images were recorded at 72 hrs at 40× enlargement. (<b>B</b>) Effect of trastuzumab on cell migration over a shorter interval (16 h) in a scratch assay. SKBr3 cells were seeded in 6-well culture plates and cultured overnight to achieve a cell density of full confluence. A scratch was made in the monolayer. Cells were then treated with trastuzumab (Tras) or control hIgG (10 µg/ml) plus epidermal growth factor (EGF, 20 ng/ml, sigma) for 16 hrs. Images were recorded at the end of 16 hrs of EGF stimulation at 40× enlargement. (<b>C</b>) Effect of trastuzumab (Tras) on cytoskeletal vinculin distribution after IF staining. SKBr3 cells were treated with trastuzumab (Tras) or control hIgG (10 µg/ml) for 48 hrs, and then subjected to IF staining as described in Methods. (<b>D</b>) Effect of trastuzumab on talin2 protein in BT474 cells. BT474 cells were treated in vitro with different concentrations of trastuzumab for 48 hrs. Total protein was prepared and subjected to Western blotting. (<b>E</b>) Quantitation of talin2 expression. The talin2 bands on immunoblots from three different experiments including the one shown in (<b>D</b>) were digitized, normalized to the levels of GAPDH, and expressed as mean levels (error bars correspond stand deviation). The talin2 expression at trastuzumab 0 concentration was set at 1. (<b>F</b>) Effect of trastuzumab on talin2 protein in SKBr3 cells. SKBr3 cells were treated with trastuzumab (Tras) or control hIgG (10 µg/ml) for 48 hrs. Total protein was prepared and subjected to Western blotting. (<b>G</b>) Effect of trastuzumab on talin2 protein in BT474 xenografts. BT474 xenografts in nude mice were treated with trastuzumab (Tras) or hIgG 1 mg/kg intraperitoneally twice a week and for 3 weeks. Total cell lysates were prepared and subjected to Western blotting.</p

miRNAs affected by trastuzumab in SKBr3 cells.

<p>miRNAs affected by trastuzumab in SKBr3 cells.</p

miRNAs affected by trastuzumab in BT474 cells.

<p>miRNAs affected by trastuzumab in BT474 cells.</p

Increased miR-194 expression inhibits breast cancer cell migration and invasion.

<p>(<b>A</b>) miR-194 expression in the stable clones of BT474 cells. BT474 cells were stably transfected with an empty pEGFP-C1 vector or pEGFP-miR-194 vector under the selection of G418. Two control clones #17 and #19 that contain empty vector and two miR-194-expressing clones #22 and #23 were established and subjected for QRT-PCR analysis. Hsa-miR-194 was purchased from ABI (Assay ID 000493). (<b>B</b>) Cell viability assay of BT474 stable cells that express miR-194 or its control vector. BT474 cells were stably transfected with empty pEGFP-C1 vector or pEGFP-miR194 construct under the selection of G418. Two control clones #17 and #19 that contain empty vector and two miR-194-expressing clones #22 and #23 were chosen to measure viability of crystal violet-stained cells on day 1, day 3 and day 5. (<b>C</b>) Effect of miR-194 precursor on cell migration in SKBr3 cells. SKBr3 cells were transiently transfected with a miR-194 precursor or a control miRNA (miR CTRL) for 48 hrs and motility was measured overnight in a Transwell assay. (<b>D</b>) Quantitation of the SKBr3 cell migration as shown in (C). <b>*</b> p<0.05 compared to miR control. (<b>E</b>) Effect of miR-194 precursor on cell invasion in SKBr3 cells. SKBr3 cells were transiently transfected with a miR-194 precursor or a control miRNA (miR CTRL) for 48 hrs and invasion measured overnight. <b>*</b> p<0.05 compared to miR control. (<b>F</b>) miR-194 expression in transiently transfected SKBr3 cells. SKBr3 cells were transiently transfected with a miR-194 precursor or a control miRNA (miR CTRL) for 48 hrs. Total RNA was extracted and subjected to QRT-PCR analysis for miR-194 expression. Hsa-miR-194 was purchased from ABI (Assay ID 000493). (<b>G</b>) Assay of cell migration in BT474 stable cells that express miR-194 or a control vector. The control clone #17 and the miR-194-expressing clone #22 were chosen to study migration. <b>*</b> p<0.05 compared to #17 control. (<b>H</b>) Cell invasion assay in BT474 stable cells that express miR-194 or its control vector. The control clone #17 and the miR-194-expressing clone #22 were chosen to study invasion. <b>*</b> p<0.05 compared to #17 control. (<b>I</b>) BT474 xenograft tumor growth in vivo. BT474 xenografts in nude mice were established with the control clone #17 and the miR-194-expressing clone #22 as described in Methods. Tumors were collected and weighed after 4 weeks. <b>*</b> p<0.05 compared to #17 control.</p

miR-194 inhibitor stimulates cell migration and blocks trastuzumab-inhibited cell migration.

<p>(<b>A</b>) Effect of miR-194 inhibitor on cell migration in SKBr3 cells. SKBr3 cells were transiently transfected with a miR-194 inhibitor or its negative control (miR inhibitor CTRL) for 48 hrs before measurement of migration overnight. <b>*</b> p<0.05 compared to the negative control. (<b>B</b>) Validation of efficacy of miR-194 inhibition. SKBr3 cells were transiently transfected with a miR-194 inhibitor or its negative control (miR inhibitor CTRL) for 48 hrs. Total RNA was prepared and miR-194 measured by QRT-PCR. <b>*</b> p<0.05 compared to miR inhibitor control. (<b>C</b>) Effect of miR-194 inhibitor on trastuzumab-inhibited cell migration. SKBr3 cells were transiently transfected with a miR-194 inhibitor or its negative control (miR inhibitor CTRL) for 16 hrs, treated with trastuzumab (Tras) or control hIgG (10 µg/ml) for 36 hrs, and motility was then measured for overnight in Transwell assays. <b>*</b> p<0.05.</p

Trastuzumab upregulates miR-194 expression in vitro and in vivo.

<p>HER2-overexpressing breast cancer cell lines BT474 (<b>A</b>) and SKBr3 (<b>B</b>) were treated with trastuzumab (Tras) or control hIgG (10 µg/ml) for 48 hrs. Total RNA was prepared and analyzed by QRT-PCR to measure miR-194. <b>*</b> p<0.05 compared to hIgG control. (<b>C</b>) Northern blot analysis of miR-194 expression. BT474 cells were treated with Tras or control hIgG (10 µg/ml) for 48 hrs. Total RNA was prepared and analyzed by Northern blotting to detect miR-194. U6 non-coding small nuclear RNA (snRNA) served as a loading control. (<b>D</b>) QRT-PCR quantitation of miR-194 expression in vivo. BT474 xenografts in miR-194 levels were measured by QRT-PCR. <b>*</b> p<0.05 compared to hIgG control. (<b>E</b>) QRT-PCR quantitation of miR-194 expression in trastuzumab sensitive or resistant cell lines. Parental SKBr3 and BT474 (trastuzumab-sensitive), and their derived resistant cells were treated with Tras or control hIgG for 48 hrs. Total RNA was prepared and analyzed by QRT-PCR to detect miR-194. <b>*</b> p<0.05 compared to hIgG control.</p

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/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