Additional file 1: of Targeting of HER3 with Functional Cooperative miRNAs Enhances Therapeutic Activity in HER2-Overexpressing Breast Cancer Cells

Figure S1. Transfection with the multi-miRNA (cluster) lentiviral vector significantly enhanced T-DM1-induced growth inhibitory effects on BT474 cells. The stable clones of BT474 cells transfected with empty vector (pCDH), pCDH-miR-125a, or pCDH-miR-205 alone or pCDH-miR-125a-miR-205 (cluster) were seeded onto 96-well plates. After 24 hrs, the 96-well plates were then placed into the IncuCyte system to measure cell proliferation in a real time. Data show growth curves of the clones in comparison with their responses to the treatment of T-DM1 (10 Îźg/ml) in the indicated time period. (TIF 2615 kb

Cladribine in combination with entinostat synergistically elicits anti-proliferative/anti-survival effects on multiple myeloma cells

<p>Cladribine (2CdA), a synthetic purine analog interfering with DNA synthesis, is a medication used to treat hairy cell leukemia (HCL) and B-cell chronic lymphocytic leukemia. Entinostat, a selective class I histone deacetylase (HDAC) inhibitor, shows antitumor activity in various human cancers, including hematological malignancies. The therapeutic potential of cladribine and entinostat against multiple myeloma (MM) remains unclear. Here we investigate the combinatorial effects of cladribine and entinostat within the range of their clinical achievable concentrations on MM cells. While either agent alone inhibited MM cell proliferation in a dose-dependent manner, their combinations synergistically induced anti-proliferative/anti-survival effects on all MM cell lines (RPMI8226, U266, and MM1.R) tested. Further studies showed that the combinations of cladribine and entinostat as compared to either agent alone more potently induced mitotic catastrophe in the MM cells, and resulted in a marked increase of the cells at G1 phase associated with decrease of Cyclin D1 and E2F-1 expression and upregulation of p21^waf−1. Apoptotic ELISA and western blot analyses revealed that the combinations of cladribine and entinostat exerted a much more profound activity to induce apoptosis and DNA damage response, evidenced by enhanced phosphorylation of histone H2A.X and the DNA repair enzymes Chk1 and Chk2. Collectively, our data demonstrate that the combinations of cladribine and entinostat exhibit potent activity to induce anti-proliferative/anti-survival effects on MM cells via induction of cell cycle G1 arrest, apoptosis, and DNA damage response. Regimens consisting of cladribine and/or entinostat may offer a new treatment option for patients with MM.</p> <p><b>Abbreviations:</b> MM, multiple myeloma; HCL, hairy cell leukemia; HDAC, histone deacetylase; Ab, antibody; mAb, monoclonal Ab; FBS, fetal bovine serum; CI, combination index; PAGE, polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay; PARP, poly(ADP-ribose) polymerase; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt</p

Induction of late-stage apoptosis and necrosis by EDC-Herceptin conjugate treatment.

<p>(<b>a</b>) OS187 cells were treated with sham, wt Herceptin (wild-type Herceptin), or EDC conjugate (EDC-Herceptin conjugate) for 48 or 72 h and then stained with annexin v and PI to determine the necrosis and apoptosis under flow cytometry. Necrosis control, freeze cells in −80°C for 5 min, and then thaw cells at 37°C. Serum starving, serum starving overnight as apoptosis control. (<b>b</b>) SKBR3 cells were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023270#pone-0023270-g003" target="_blank">Figure 3</a>, followed by PI, annexin v staining, and flow cytometry analysis.</p

Induction of Herceptin-resistant tumor cell death by EDC-Herceptin conjugate.

<p>Herceptin-resistant tumor cells HR20 were treated with wild-type Herceptin or EDC conjugate at low concentration (20 µg/mL) or high concentration (80 µg/mL) for 72 h, and cell survival rates were analyzed using MTT assay. (<b>a</b>) Cell survival rate at the end of the described treatment. (<b>b</b>) Her2 expression level analyzed by flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023270#pone-0023270-g001" target="_blank">Figure 1</a>. (<b>c</b>) Detection of cell death frequency following different treatments. Cells were treated with sham, wt low (wild-type Herceptin 20 µg/mL), EDC low (EDC-Herceptin conjugate 20 µg/mL), wt high (wild-type Herceptin 80 µg/mL), or EDC high (EDC-Herceptin conjugate 80 µg/mL). Live and dead cells were analyzed using a live/dead cell staining kit (see detail in Methods). Green fluorescence stained cells, live cells; Red florescence stained cells, dead and dying cells. ***, <i>P</i><0.001.</p

Treatment of Her2-positive or Her2-negative tumor cells by EDC-Herceptin conjugate.

<p>(<b>a</b>) Comparison of cell survival rates between wild-type Herceptin and EDC-Herceptin conjugate treatments in Her2-positive (OS187, CCH.OS.D, and SKBR3) and Her2-negative (Col and MDA-MB-468) cell lines. MTT assay was used after cells were treated with the described antibody at low concentration (20 µg/mL) or high concentration (80 µg/mL) for 72 h. (<b>b</b>) Her2 expression analysis in tested cell lines using flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023270#pone-0023270-g001" target="_blank">Figure 1</a>. *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

Analysis of cell death mechanism.

<p>(<b>a</b>) Active caspase analysis. OS187 and SKBR3 cells were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023270#pone-0023270-g002" target="_blank">Figure 2</a>. Caspase-3, -8, and -9 activity were tested by CaspGLOW fluorescein active caspase staining kits. Red dot line, sham; black dash line, wild-type Herceptin (80 µg/mL); black solid line, EDC-Herceptin conjugate (80 µg/mL). (<b>b</b>). Effect of caspase inhibitors on EDC conjugate-induced cell death. Z-DEVD-FMK, caspase 3 inhibitor. Z-IETD-FMK, caspase 8 inhibitor. Z-LEHD-FMK caspase 9 inhibitor. Z-VAD-FMK, caspase inhibitor. OS187 cells were treated with sham, EDC Herceptin conjugate 20 µg/mL or EDC Herceptin conjugate 20 µg/mL plus different caspase inhibitors at 30 µmol/L. (<b>c</b>) Effect of necrosis inhibitors on EDC-Herceptin conjugate-induced cell death. Necrostatin-1 (Nec-1), programmed cell necrosis inhibitor. OS187/SKBR3 cells were treated with sham, EDC-Herceptin conjugate 20/80 µg/mL, or EDC-Herceptin conjugate 20/80 µg/mL plus Nec-1 at 30 µmol/L. N = 5. *, <i>P</i><0.05; **, <i>P</i><0.01.</p

Suppression of Her2-positive tumor cell survival by EDC-Herceptin conjugate.

<p>(<b>a</b>) Gel analysis of the Herceptin conjugate. Analysis of Herceptin and Herceptin conjugates at 6% SDS-PAGE without reducing reagent DTT. Lane 1, wild-type Herceptin; lane 2, EDC-Herceptin conjugate; lane 3, SMCC-Herceptin conjugate; lane 4, molecular weight marker. (<b>b</b>) Reduction of Her2-positive tumor cell survival by EDC-Herceptin conjugate but not by SMCC-Herceptin conjugate. OS187 and CCH.OS.D cells were treated with the indicated molecules at 20 µg/mL for 72 h. MTT assay was used to determine the cell survival rate. (<b>c</b>) Her2 high expression cell lines OS187 and CCH.OS.D. Cells were stained with isotype control or PE-Her2 antibody. Her2 expression level was determined by using flow cytometry. Red dotted line, isotype control; blue line, Her2. *, <i>P</i><0.05; **, <i>P</i><0.01.</p

Metformin attenuates transforming growth factor beta (TGF-β) mediated oncogenesis in mesenchymal stem-like/claudin-low triple negative breast cancer

<p>Mesenchymal stem-like/claudin-low (MSL/CL) breast cancers are highly aggressive, express low cell-cell adhesion cluster containing claudins (CLDN3/CLDN4/CLDN7) with enrichment of epithelial-to-mesenchymal transition (EMT), immunomodulatory, and transforming growth factor-β (TGF-β) genes. We examined the biological, molecular and prognostic impact of TGF-β upregulation and/or inhibition using <i>in vivo</i> and <i>in vitro</i> methods. Using publically available breast cancer gene expression databases, we show that upregulation and enrichment of a TGF-β gene signature is most frequent in MSL/CL breast cancers and is associated with a worse outcome. Using several MSL/CL breast cancer cell lines, we show that TGF-β elicits significant increases in cellular proliferation, migration, invasion, and motility, whereas these effects can be abrogated by a specific inhibitor against TGF-β receptor I and the anti-diabetic agent metformin, alone or in combination. Prior reports from our lab show that TNBC is exquisitely sensitive to metformin treatment. Mechanistically, metformin blocks endogenous activation of Smad2 and Smad3 and dampens TGF-β-mediated activation of Smad2, Smad3, and ID1 both at the transcriptional and translational level. We report the use of ID1 and ID3 as clinical surrogate markers, where high expression of these TGF-β target genes was correlated to poor prognosis in claudin-low patients. Given TGF-β's role in tumorigenesis and immunomodulation, blockade of this pathway using direct kinase inhibitors or more broadly acting inhibitors may dampen or abolish pro-carcinogenic and metastatic signaling in patients with MCL/CL TNBC. Metformin therapy (with or without other agents) may be a heretofore unrecognized approach to reduce the oncogenic activities associated with TGF-β mediated oncogenesis.</p

FRAT1 depletion decreases tumorigenicity in nude mice.

<p>(A) BALB/c-nu mice were injected subcutaneously with 1×10⁷ of U251, U251-neo, or U251-NC control cells; or U251-S FRAT1 knockdown cells. Representative tumor formation was photographed 40 days after injection. (B) Tumor sizes were determined by measuring the tumor volume every five days from 5 to 40 days after injection. (C) Average tumor weights of mice 40 days after injection are shown. Values represent means±SD obtained from three independent experiments. (D) Immunohistochemical analysis of FRAT1 expression in tumors in nude mice 40 days following injection.</p

RNAi-mediated knockdown of FRAT1 inhibits growth of U251 cells in vitro.

<p>Cell viability was measured using a MTT assay. Cell growth curves were determined by absorbance at 490 nm. *, P<0.01 for U251-S relative to each of the three control lines.</p