Bone Morphogenetic Protein Type I Receptor Antagonists Decrease Growth and Induce Cell Death of Lung Cancer Cell Lines

<div><p>Bone morphogenetic proteins (BMPs) are highly conserved morphogens that are essential for normal development. BMP-2 is highly expressed in the majority of non-small cell lung carcinomas (NSCLC) but not in normal lung tissue or benign lung tumors. The effects of the BMP signaling cascade on the growth and survival of cancer cells is poorly understood. We show that BMP signaling is basally active in lung cancer cell lines, which can be effectively inhibited with selective antagonists of the BMP type I receptors. Lung cancer cell lines express alk2, alk3, and alk6 and inhibition of a single BMP receptor was not sufficient to decrease signaling. Inhibition of more than one type I receptor was required to decrease BMP signaling in lung cancer cell lines. BMP receptor antagonists and silencing of BMP type I receptors with siRNA induced cell death, inhibited cell growth, and caused a significant decrease in the expression of inhibitor of differentiation (Id1, Id2, and Id3) family members, which are known to regulate cell growth and survival in many types of cancers. BMP receptor antagonists also decreased clonogenic cell growth. Knockdown of Id3 significantly decreased cell growth and induced cell death of lung cancer cells. H1299 cells stably overexpressing Id3 were resistant to growth suppression and induction of cell death induced by the BMP antagonist DMH2. These studies suggest that BMP signaling promotes cell growth and survival of lung cancer cells, which is mediated through its regulation of Id family members. Selective antagonists of the BMP type I receptors represents a potential means to pharmacologically treat NSCLC and other carcinomas with an activated BMP signaling cascade.</p></div

Inhibition of type I BMP receptors in lung cancer cell lines induces cell death.

<p>(<b>A–B</b>) H1299 and A549 cultured in DMEM 5% FCS were treated with DMSO or a BMP receptor antagonist (10 µM Dorsomorphin or 1 µM of selective antagonist). The percentage of cells that take up ethidium bromide was then determined. The data is reported as the mean of at least 3 independent experiments. (<b>C</b>) H1299 cells cultured in DMEM 5% FCS were treated with DMSO, 1 µM and μ5 M of DMH2 for 7 days, stained with Typan Blue, and cell counts performed. Data represents the mean of 4 experiments reported as the percent dead cells. (<b>D</b>) H1299 cells cultured in SFM were treated with DMSO or 1 µM of DMH2 for 7 days, stained with Typan Blue, and cell counts performed. Data represents the mean of 5 experiments reported as percent dead cells. (<b>E</b>) Cell death was determined using flow cytometry detecting uptake of an amine-reactive fluorescent dye in H1299 cells treated with DMS0 or DMH2 for 4 days. Data represent the mean of 3 independent experiments. (<b>F</b>) The H1299 cells were transfected with control siRNA and siRNA targeting alk2, alk3, and alk6. After 2 days the percentage of cells staining for ethidium bromide was determined. The data is reported as the mean of 6 independent experiments.</p

DMH1 treatment does not specifically induce expression of endoderm or ectoderm markers.

<p>DMH1 treatment does not have a statistically significant effect on definitive endoderm markers FoxA2 and Sox17, or ectoderm progenitor marker Nestin. The data show DM treatment results in a statistically significant reduction in FoxA2 expression on Day 4, and a general delay in Sox17 expression. There is no significant increase in the expression of Nestin after DM treatment. Results are presented as the average of three independent experiments. Expression levels shown are normalized to Day 0 expression levels. Error bars denote the S.E.M. P-value is calculated using two-tailed Student’s t-test. DMSO is the vehicle control. DM is dorsomorphin treatment. DMH1 is dorsomorphin homologue 1.</p

Antagonizing BMP type I receptors decreases cell growth, proliferation, and clonogenicity of lung cancer cell lines.

<p>(<b>A</b>) A549 and H1299 cells cultured in DMEM 5% FCS were treated with DMSO, 10 µM Dorsomorphin, 1 µM DMH1, 1 µM DMH2, or 1 µM LDN for 7 days and cell counts performed. (<b>B</b>) H1299 cells cultured in SFM were treated with 1 µM DMSO or 1 µM DMH2 for 7 days and cell counts performed. (<b>C</b>) BrdU incorporation of H1299 cells treated with DMSO or 1 µM or 5 µM DMH2 for 24 and 48 hours. (<b>D</b>) BrdU incorporation of H1299 cells transfected with siRNA targeting of all type I BMP receptors or siRNA control. (<b>C–D</b>) Data is the mean of 3 experiments in triplicate reported as the percent of control treated cells. (<b>E–G</b>) Colony growth of A549 and H1299 cells treated with 1 µM DMSO or 1 µM of selective BMP receptor antagonist. (<b>E</b>) Photograph of a representative experiment. (<b>F–G</b>) The data shows the mean of at least 3 independent experiments reported as the percent of control. (<b>G</b>) DMH1 decreases anchorage independent growth of lung cancer cell lines. A549 and H1299 cells in soft agar were treated with 1 µM DMS0 or 1 µM DMH1 for 2 weeks and the number of colonies counted. The data shown is the mean of 3 independent experiments reported as the percent of control.</p

Lung cancer cell lines secrete BMP2 and knockdown of BMP2 decreases BMP signaling and induces cell death.

<p>(<b>A</b>) A BMP2 Elisa was performed on the DMEM 5% FCS and SFM in the absence of cells (medium). A BMP2 Elisa was performed on the DMEM 5% FCS and SFM cell culture medium containing A549 and H1299 cells for 48 hours. Experiments represent the mean of at least 5 experiments performed in triplicate. (<b>B</b>) BMP antagonists decrease BMP signaling of lung cancer cells cultured in SFM. H1299 cells cultured in SFM were treated with 1 µM DMSO, 1 µM of DMH2 or 1 µM of LDN for 48 hours and Western blot analysis performed. (<b>C</b>) Quantitative RT-PCR for BMP2 expression 48 hours after transfecting H1299 cells with control siRNA or siRNA targeting BMP2. Data represents the percent of control of the average of 4 independent experiments. (<b>D</b>) Western blot analysis of H1299 cells in SFM 48 hours after being transfected with control siRNA or siRNA targeting BMP2. BMP2 knockdown causes a decrease in expression of pSmad 1/5 and Id1. (<b>E</b>) Knockdown of BMP2 induces cell death. H1299 cells in SFM were transfected with control siRNA or siRNA targeting BMP2. After 48 hours the percentage of dead cells was determined by ethidium bromide staining. The data represents the mean of 4 independent experiments performed in triplicate.</p

Low BryT-expressing fraction produces beating EB after Wnt inhibition.

<p>Each table is data from a single experiment showing the percentage of beating aggregates that formed after GFP– fractions were treated with either DMSO control or 1 µM XAV939 to inhibit Wnt signaling. The DMH1-treated GFP– fractions typically form beating aggregates 6 to 8 days after XAV939 addition (Day 10 to Day 12 after initial induction). The percentages increase over time and the efficiency of beating aggregate formation varies with each FACS. The percentages were calculated from the number of beating aggregates and the number of total aggregates for each condition.</p

Id1 and Id3 regulate cell growth and survival of lung cancer cells.

<p>(<b>A</b>) Western blot analysis for Id1 and Id3 48 hours after H1299 cells were transfected with control siRNA and siRNA targeting Id1 or Id3. (<b>B</b>) Quantitative RT-PCR for Id1 and Id3 after H1299 cells were transfected with control siRNA and siRNA targeting Id1 or Id3<b>.</b> Data represents the percent control of the mean of 2 experiments. (<b>C</b>) H1299 cells were transfected with control siRNA or siRNA targeting Id1 or Id3. After 48 hours the percentage of cells staining for ethidium bromide was determined. The data is reported as the mean of 4 independent experiments. (<b>D–F</b>) H1299 cells were transfected with control siRNA and siRNA targeting Id1 or Id3. After 7 days the cells were stained with Trypan Blue and the number of alive and dead cells was determined. The data represents the percent change from siRNA control of (<b>D</b>) alive or (<b>F</b>) dead cells. (<b>E</b>) Represents the percentage of cells that were dead. The data represents the mean of 4 independent experiments.</p

Forced expression of Id3 prevents growth suppression and cell death induced by DMH2.

<p>H1299 cells were stably transfected with Id1 and Id3 expression vectors or the insertless vector. (<b>A–B</b>) Western blot analysis showing increased expression of (<b>A</b>) Id1 or (<b>B</b>) Id3 in the transfected cell line. (<b>C–D</b>) H1299/Id1 and H1299/Id3 cells were treated with 1 µM DMSO or 1 µM DMH2 for 7 days and the percent alive and dead cells determined. (<b>C</b>) DMH2 caused growth suppression of vector control and H1299/Id1 cells but not H1299/Id3 cells. (<b>D</b>) DMH2 induced cell death in the vector control cells (H/con) but not in the H1299/Id3 cells (H/Id3). The data represents the mean of at least 3 experiments reported as the percent of control treated cells. (<b>E</b>) DMH2 deceases cell growth of immortalized normal human bronchial epithelial cells (HBEC) but not human aortic endothelial cells (HAEC). Cell lines growing in SFM were treated with 1 µM DMSO or 1 µM DMH2 for 7 day and cell counts performed. Data represents the mean of at least 3 experiments reported as the percent of control treated cells. (<b>F</b>) Western blot analysis showing higher expression of pSmad 1/5 and Id1 in HBEC compared to HAEC. 1 µM of DMH2 for 48 hours decreased expression of pSmad 1/5 and ID1 in HBEC but not HAEC.</p

FACS analysis confirms BryT induction by DMH1 and limited BryT induction by DM.

<p>Mouse embryonic stem cells containing a recombinant BryT-GFP reporter were induced as previously described and analyzed using FACS after 4 days of treatment with various compounds. (Top) The percentages of GFP+ cells calculated from at least 10,000 parental cells are shown. The range shows the variability of total GFP+ cells for the experiments. The data show DM treatment yields fewer GFP+ cells than DMH1-treated cells. At least two independent experiments are presented as the mean. SD is standard deviation. (Bottom) BryT expression levels in the GFP+ fractions are confirmed using rt-PCR. GFP+ cells also show increased Mesp1 and Isl1 expression, which indicates a transition to mesoderm and cardiac lineage commitment. Rt-PCR results are presented as the average of three independent experiments. Expression levels shown are normalized to Day 0 expression levels. Error bars denote the S.E.M. DMSO is the vehicle control. DM is dorsomorphin treatment. DMH1 is dorsomorphin homologue 1.</p

DMH1, a Novel BMP Small Molecule Inhibitor, Increases Cardiomyocyte Progenitors and Promotes Cardiac Differentiation in Mouse Embryonic Stem Cells

<div><p>The possibility of using cell-based therapeutics to treat cardiac failure has generated significant interest since the initial introduction of stem cell-based technologies. However, the methods to quickly and robustly direct stem cell differentiation towards cardiac cell types have been limited by a reliance on recombinant growth factors to provide necessary biological cues. We report here the use of dorsomorphin homologue 1 (DMH1), a second-generation small molecule BMP inhibitor based on dorsomorphin, to efficiently induce beating cardiomyocyte formation in mouse embryonic stem cells (ESCs) and to specifically upregulate canonical transcriptional markers associated with cardiac development. DMH1 differs significantly from its predecessor by its ability to enrich for pro-cardiac progenitor cells that respond to late-stage Wnt inhibition using XAV939 and produce secondary beating cardiomyocytes. Our study demonstrates the utility of small molecules to complement existing in vitro cardiac differentiation protocols and highlights the role of transient BMP inhibition in cardiomyogenesis.</p> </div