The combination of gefitinib and RAD001 inhibits growth of HER2 overexpressing breast cancer cells and tumors irrespective of trastuzumab sensitivity

<p>Abstract</p> <p>Background</p> <p>HER2-positive breast cancers exhibit high rates of innate and acquired resistance to trastuzumab (TZ), a HER2-directed antibody used as a first line treatment for this disease. TZ resistance may in part be mediated by frequent co-expression of EGFR and by sustained activation of the mammalian target of rapamycin (mTOR) pathway. Here, we assessed feasibility of combining the EGFR inhibitor gefitinib and the mTOR inhibitor everolimus (RAD001) for treating HER2 overexpressing breast cancers with different sensitivity to TZ.</p> <p>Methods</p> <p>The gefitinib and RAD001 combination was broadly evaluated in TZ sensitive (SKBR3 and MCF7-HER2) and TZ resistant (JIMT-1) breast cancer models. The effects on cell growth were measured in cell based assays using the fixed molar ratio design and the median effect principle. <it>In vivo </it>studies were performed in Rag2M mice bearing established tumors. Analysis of cell cycle, changes in targeted signaling pathways and tumor characteristics were conducted to assess gefitinib and RAD001 interactions.</p> <p>Results</p> <p>The gefitinib and RAD001 combination inhibited cell growth <it>in vitro </it>in a synergistic fashion as defined by the Chou and Talalay median effect principle and increased tumor xenograft growth delay. The improvement in therapeutic efficacy by the combination was associated <it>in vitro </it>with cell line dependent increases in cytotoxicity and cytostasis while treatment <it>in vivo </it>promoted cytostasis. The most striking and consistent therapeutic effect of the combination was increased inhibition of the mTOR pathway (<it>in vitro </it>and <it>in vivo</it>) and EGFR signaling <it>in vivo </it>relative to the single drugs.</p> <p>Conclusions</p> <p>The gefitinib and RAD001 combination provides effective control over growth of HER2 overexpressing cells and tumors irrespective of the TZ sensitivity status.</p

Asymmetric Cell Divisions Sustain Long-Term Hematopoiesis from Single-sorted Human Fetal Liver Cells

Hematopoietic stem cells (HSCs) in adult marrow are believed to be derived from fetal liver precursors. To study cell kinetics involved in long-term hematopoiesis, we studied single-sorted candidate HSCs from fetal liver that were cultured in the presence of a mixture of stimulatory cytokines. After 8–10 d, the number of cells in primary cultures varied from <100 to >10,000 cells. Single cells in slow growing colonies were recloned upon reaching a 100–200 cell stage. Strikingly, the number of cells in subclones varied widely again. These results are indicative of asymmetric divisions in primitive hematopoietic cells in which proliferative potential and cell cycle properties are unevenly distributed among daughter cells. The continuous generation of functional heterogeneity among the clonal progeny of HSCs is in support of intrinsic control of stem cell fate and provides a model for the long-term maintenance of hematopoiesis in vitro and in vivo

Induction of Autophagy Is an Early Response to Gefitinib and a Potential Therapeutic Target in Breast Cancer

Gefitinib (Iressa®, ZD1839) is a small molecule inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase. We report on an early cellular response to gefitinib that involves induction of functional autophagic flux in phenotypically diverse breast cancer cells that were sensitive (BT474 and SKBR3) or insensitive (MCF7-GFPLC3 and JIMT-1) to gefitinib. Our data show that elevation of autophagy in gefitinib-treated breast cancer cells correlated with downregulation of AKT and ERK1/2 signaling early in the course of treatment. Inhibition of autophagosome formation by BECLIN-1 or ATG7 siRNA in combination with gefitinib reduced the abundance of autophagic organelles and sensitized SKBR3 but not MCF7-GFPLC3 cells to cell death. However, inhibition of the late stage of gefitinib-induced autophagy with hydroxychloroquine (HCQ) or bafilomycin A1 significantly increased (p&lt;0.05) cell death in gefitinib-sensitive SKBR3 and BT474 cells, as well as in gefitinib-insensitive JIMT-1 and MCF7-GFPLC3 cells, relative to the effects observed with the respective single agents. Treatment with the combination of gefitinib and HCQ was more effective (p&lt;0.05) in delaying tumor growth than either monotherapy (p&gt;0.05), when compared to vehicle-treated controls. Our results also show that elevated autophagosome content following short-term treatment with gefitinib is a reversible response that ceases upon removal of the drug. In aggregate, these data demonstrate that elevated autophagic flux is an early response to gefitinib and that targeting EGFR and autophagy should be considered when developing new therapeutic strategies for EGFR expressing breast cancers

Gefitinib inhibits cell growth and induces appearance of MDC-labeled organelles in breast cancer cells.

<p>(<b>A</b>) Example images obtained with IN Cell 1000 showing SKBR3 cells treated for 72 h with 10 µM gefitinib and stained <i>in </i><i>situ</i> with DRAQ5 (stains nuclei in viable and dead cells), ETH (stains nuclei in dead cells with compromised plasma membrane) and MDC (stains acidic organelles). Based on differential staining and morphological features image recognition software identifies different cell populations. Left image: nuclear imaging masks shown in blue and red indicate viable and dead cells, respectively; cytoplasm of viable cells with MDC-labeled organelles is shown in yellow and cytoplasm of cells without MDC-labeled organelles is shown in light blue. Middle image: magnification of an area, as marked in the left image, showing transparent imaging masks outlining nuclei, cytoplasm and MDC-labeled organelles, as recognized by the HCA Investigator software; DRAQ5 staining is shown in blue, ETH staining is shown in red and MDC staining is shown in green. Right image: a high magnification image showing MDC-labeled organelles outlined in yellow within the cells’ cytoplasm; some of the MDC-labeled structures, indicated by the red arrows, represent multiple closely grouped organelles. (<b>B</b>) Representative images of indicated cells cultured in the presence of 0.5% DMSO (vehicle) or 10 µM gefitinib for 24 h (BT474) or 72 h (SKBR3, JIMT-1, MCF7-GFPLC3) stained <i>in </i><i>situ</i> with DRAQ5 (blue), ETH (red) and MDC (green). Images in (<b>A</b>) and (<b>B</b>) were pseudo-colored and overlaid using the Investigator software. (<b>C</b>) Quantitation of different cell populations and autophagic organelles in BT474, SKBR3, JIMT-1 and MCF7-GFPLC3 cells by HCA. Cells were treated with vehicle or increasing concentrations of gefitinib for the indicated time. Numbers of viable cells in culture are normalized to vehicle-treated controls. Dead cells are shown as a percent difference in the content of dead (ETH-positive) cells between drug-treated minus vehicle-treated cultures. The proportion of viable cells with MDC-labeled organelles (puncta) in culture is shown as a percent difference in cells with >1 MDC-labeled organelle between drug-treated minus vehicle-treated cultures. Each data point represents a mean±SD from 3 replicate wells. HCA screenings were repeated 2 - 3 times for each cell type with consistent results; representative experiments are shown. </p

Effects of siRNA mediated EGFR silencing on downstream signaling and autophagy.

<p>(<b>A</b>) Following a transfection with EGFR siRNA, SKBR3 cells were treated for 72 h with vehicle or 5 µM gefitinib. (<b>B</b>) Following a double knockdown with the EGFR siRNA MCF7-GFPLC3 cells were treated with vehicle or 4 µM gefitinib for 24 h. Representative experiments are shown.</p

3-MA sensitized SKBR3 but not MCF7-GFPLC3 cells to cell death in the presence of gefitinib.

<p>Flow cytometric analysis of apoptosis in SKBR3 (<b>A</b> and <b>B</b>) and MCF7-GFPLC3 (<b>C</b> and <b>D</b>) cells treated for 72 h with vehicle or 10 µM gefitinib (Gef), in the absence or presence of 5 mM 3-MA. Camptothecin (CPT) at 5 µM was used as an inducer of apoptosis (positive control). (<b>A</b> and <b>C</b>) Analysis of the sub-G₀/G₁ apoptotic cell fraction. The inserted histograms show representative DNA profiles of cells treated with the indicated agents where a sub-G₀/G₁ cell fraction is indicated with a marker. Arrows in (<b>C</b>) indicate S-G₂/M cell cycle block. (<b>B</b> and <b>D</b>) Analysis of apoptosis in cells stained with Annexin V-Alexa647 and PI. The inserted representative dot plots show distribution of cell populations treated with the indicated agents where apoptotic Annexin V-positive cells are marked as “A” in a rectangular region. Bar graphs represent the data (mean±SD) from 3 independently stained samples and show fold change relative to the vehicle-treated controls expressed as 1. Asterisks indicate a statistically significant difference (p<0.05) between cells treated with the vehicle and indicated agents. A double asterisk indicates a statistically significant difference (p<0.05) between cells treated with gefitinib in the presence of 3-MA and single-agent treated cells in addition to a statistically significant difference when compared to the vehicle-treated cells. Representative experiments are shown.</p

Gefitinib induces autophagic flux.

<p>(<b>A</b> - <b>D</b>) Autophagic flux assays performed in SKBR3 cells. (<b>A</b>) Western blot analysis of p62 expression in lysates derived from cells treated for 3 h with vehicle (0 µM gefitinib) or increasing concentrations of gefitinib (Gef) (<b>B</b>) Western blot analysis of LC3 levels in lysates derived from cells treated for 3 h with increasing concentrations of gefitinib in the absence or presence of 5 nM bafilomycin A1 (BAF). (<b>C</b>) HCA of TOA in cells treated for 72 h with increasing concentrations of gefitinib in the absence or presence of 10 mM 3-MA added for the last 3 h of treatment. The results are normalized to the vehicle control expressed as 1. Each bar represents the mean±SD from 3 replicate wells. Asterisks indicate statistically significant differences (p<0.05) between cells treated with gefitinib or 3-MA alone and cells treated with 3-MA in the presence of gefitinib. The results shown are representative of two experiments. (<b>D</b>) Western blot analysis of LC3-I and LC3-II levels in lysates derived from cells treated for 24 h with vehicle, 5 µM gefitinib, 5 mM 3-MA and the combination of gefitinib and 3-MA at the corresponding concentrations. Representative blots in (<b>A</b>), (<b>B</b>) and (<b>D</b>) are shown. (<b>E</b> - <b>G</b>) Autophagic flux assays performed in MCF7-GFPLC3 cells. (<b>E</b>) Western blot analysis of autophagic markers in lysates derived from cells treated for 18 h with increasing concentrations of gefitinib. Cleaved GFP is marked as GFP. (<b>F</b>) Western blot analysis of cleaved GFP levels in lysates derived from cells treated with vehicle or 10 µM gefitinib in the absence or presence of 10 mM 3-MA for 3 h (top panel), in the absence or presence of 50 nM bafilomycin A1 (BAF) for 24 h (middle panel), and in the absence or presence of 10 mg/ml lysosomal inhibitors (LYI; pepstatin A and E-64d) added for the last hour of treatment (bottom panel). Tubulin was used as loading control. Representative blots in (<b>E</b>) and (<b>F</b>) are shown. (<b>G</b>) HCA data showing the average GFPLC3 TOA/cell in MCF7-GFPLC3 cells treated for 3 h with increasing concentrations of gefitinib in the absence or presence of indicated autophagy inhibitors added for the last hour of treatment. The results are normalized to vehicle control expressed as 1. Each data point represents the mean±SD from 3 replicate wells and the results shown are representative of two experiments. 3-MA was used at 5 mM, bafilomycin A1 (BAF) was used at 5 nM and LYI were used at 10 µg/ml. Asterisks indicate statistically significant differences (p<0.05) between cells treated with gefitinib or autophagy inhibitors alone and cells treated with autophagy inhibitors in the presence of gefitinib. </p