Cas proteins negatively regulate E-cadherin expression in MCF7 cells.

<p><b>A</b>. Western analysis of MCF7 cells transfected with plasmids including vector pcDNA-HA (-), pcDNA-HA-NEDD9 (N), pcDNA-HA-BCAR1 (B), or pcDNA-HA-NEDD9 and pcDNA-HA-BCAR1 (NB). <b>B</b>. Western analysis of MCF7 transfected with siRNAs including scrambled control (scr), or targeting NEDD9 (siN), BCAR1 (siB) and both (siNB), probed with antibodies indicated. <b>C</b>. Graph represents total levels of E-cadherin normalized to β-actin. *, P<0.01, **, P<0.001. Error bars represent SE. Additional siRNA experiments (not shown) were performed with alternative siRNA oligonucleotides targeting NEDD9 and BCAR1: although knockdown was not as efficient, qualitatively similar results were obtained in regard to E-cadherin expression.</p

Cas proteins negatively regulate association of E-cadherin with cell junctions.

<p><b>A</b>. qRT-PCR of mRNA isolated from MCF7 cells transfected with plasmids including vector pcDNA-HA (-), pcDNA-HA-NEDD9 (N), pcDNA-HA-BCAR1 (B), or pcDNA-HA-NEDD9 and pcDNA-HA-BCAR1 (NB). <b>B</b>. MCF7 transfected with siRNAs including scrambled control (SCR), or targeting NEDD9 (siN), BCAR1 (siB) and both (siNB), were stained for E-cadherin (red), and DAPI (blue). <b>C</b>. MCF7 cells transfected as in <b>A</b> and stained for E-cadherin (red), HA-fused proteins (green) and DAPI (blue). Arrows indicate cell-cell contacts of transfected cells. <b>D</b>. MCF7 cells transfected with plasmids including vector pcDNA-GFP, pcDNA-GFP-NEDD9, pcDNA-GFP-BCAR1, or pcDNA-GFP-NEDD9 and pcDNA-GFP-BCAR1 (green) were stained for β-catenin (red), and DAPI (blue). Arrowheads point to β-catenin localization at the focal adhesions. Scale bar in <b>B–D</b>, 20 µm. <b>E</b>. Immunofluorescence demonstrating that β-catenin localizes to focal adhesions in MCF7 cells expressing pcDNA-GFP-NEDD9 and pcDNA-GFP-BCAR1 (green). β-catenin (red) and the focal adhesion protein paxillin (blue) are indicated. Scale bar, 20 µm. Bottom panel represents magnifications of indicated areas from boxes.</p

Cas proteins activate SRC to downregulate E-cadherin expression.

<p><b>A</b>, <b>B</b>. Western analysis (<b>A</b>) of whole cell lysates prepared from MCF-7 cells overexpressing pcDNA vector alone (-), or HA-tagged BCAR1 (B), NEDD9 (N), or both (NB), and treated with dasatinib versus vehicle. Quantification of results comparing levels of E-cadherin, normalized to β-actin, within treated and, separately, untreated groups from 4 independent experiments, is also shown (<b>B</b>). Src Y⁴¹⁶ phosphorylation reflects activity state of kinase. P values reflect the difference between the vehicle and drug treatment condition, for each transfected protein indicated; *, P<0.01, **, P<0.001, ns, non-significant. <b>C</b>. Experiment as in <b>A</b> performed with PP2. Note, SRC inhibition was not as complete as in <b>A</b> in these experiments, because at higher PP2 concentrations extensive cell death was observed, probably due to the broader spectrum of PP2 versus dasatinib targets. <b>D</b>, <b>E</b>. Experiment as in <b>A</b>, <b>B</b>, but in cells treated with Y27632 versus vehicle. Phosphorylation of the downstream p160ROCK target MYPT1 was assessed in parallel to confirm complete inhibition of p160ROCK (not shown). *, P<0.001, **, P<0.01 ***, P<0.0001, ns, non-significant. Error bars represent SE.</p

Nedd9<i>^−/−</i> mammary tumors have increased E-cadherin at cell junctions.

<p><b>A</b>, <b>B</b>. Immunohistochemical evaluation of MMTV-PyVT;<i>Nedd9^−/−</i> and MMTV-PyVT <i>Nedd9^+/+</i> mammary tumors for E-cadherin (<b>A</b>) and β-catenin (<b>B</b>) protein expression and localization. Insets (thick lines) are magnifications of indicated areas (thin lines). Three representative independently arising tumors are shown for each genotype. Scale bar, 100 µm. <b>C</b>. Western analysis of total lysates isolated from 9 independent MMTV-PyVT;<i>Nedd9^−/−</i> and MMTV-PyVT <i>Nedd9^+/+</i> mammary tumors and from MCF7 cells were probed for content of E-cadherin and β-catenin. β-actin served as loading control.</p

Fractionation indicates Cas proteins decrease E-cadherin association with the insoluble fraction.

<p>MCF7 cells were transfected as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022102#pone-0022102-g002" target="_blank"><b>Figure 2A and B</b></a>, fractionated to insoluble (<b>A</b>) and soluble (<b>B</b>) fractions, and then analyzed for expression of E-cadherin. Graphs represent E-cadherin normalized to β-actin in each fraction calculated from 3 independent experiments. Immunoblot represents a typical experiment. *, P<0.05, **, P<0.01, ***, P<0.001. Error bars represent SE.</p

Cas proteins increase lysosomal colocalization of E-cadherin.

<p><b>A</b>. Immunofluorescence assessing E-cadherin localization, with lysosomes visualized with LAMP-1 (green) in cells overexpressing GFP-BCAR1 (B) and GFP-NEDD9 (N), indicated in blue, after treatment with cloroquine. E-cadherin is shown in red. Scale bar, 20 µm. Arrows indicate colocalized E-cadherin and LAMP-1. <b>B–E</b>. Quantification of western analysis and representative results of whole cell lysates prepared from MCF-7 cells transfected with either the vector pcDNA-GFP, pcDNA-HA-NEDD9 (N), pcDNA-HA-BCAR1 (B), or pcDNA-HA-NEDD9 and pcDNA-HA-BCAR1 (NB), and treated with <b>B</b>. vehicle (DMSO), <b>C</b>. ammonium chloride (NH₃Cl), <b>D</b>. monensin or <b>E</b>. chloroquine, to inhibit lysosomal action. Graphs represents total levels of E-cadherin normalized to β-actin, calculated from more than 4 independent experiments. P values were calculated to compare levels of E-cadherin in vector-transfected cells versus cells transfected with NEDD9, BCAR1 or both within each drug treatment group; *, P<0.05, **, P<0.01, n.s., non-significant. Error bars represent SE.</p

Treatment with SRC inhibitors reverses CAS-induced downregulation of E-cadherin.

<p>Immunofluorescence of MCF7 cells expressing vector pcDNA-GFP, pcDNA-GFP-NEDD9, pcDNA-GFP-BCAR1, or pcDNA-GFP-NEDD9 and pcDNA-GFP-BCAR1 (transfected cells shown in green) and treated with vehicle or PP2. Arrows indicate presence of E-cadherin (shown in red) at cell junctions in dasatinib or PP2-treated cells expressing vector, BCAR1 and/or NEDD9, but absence of E-cadherin in similarly transfected cells treated with vehicle. Scale bar, 20 µm.</p

Tumor-targeted SN38 inhibits growth of early stage non-small cell lung cancer (NSCLC) in a KRas/p53 transgenic mouse model

<div><p>Non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide, with a 5-year survival of only ~16%. Potential strategies to address NSCLC mortality include improvements in early detection and prevention, and development of new therapies suitable for use in patients with early and late stage diagnoses. Controlling the growth of early stage tumors could yield significant clinical benefits for patients with comorbidities that make them poor candidates for surgery: however, many drugs that limit cancer growth are not useful in the setting of long-term use or in comorbid patients, because of associated toxicities. In this study, we explored the use of a recently described small molecule agent, STA-8666, as a potential agent for controlling early stage tumor growth. STA-8666 uses a cleavable linker to merge a tumor-targeting moiety that binds heat shock protein 90 (HSP90) with the cytotoxic chemical SN38, and has been shown to have high efficacy and low toxicity, associated with efficient tumor targeting, in preclinical studies using patient-derived and other xenograft models for pancreatic, bladder, and small cell lung cancer. Using a genetically engineered model of NSCLC arising from induced mutation of KRas and knockout of Trp53, we continuously dosed mice with STA-8666 from immediately after tumor induction for 15 weeks. STA-8666 significantly slowed the rate of tumor growth, and was well tolerated over this extended dosing period. STA-8666 induced DNA damage and apoptosis, and reduced proliferation and phosphorylation of the proliferation-associated protein ERK1/2, selectively in tumor tissue. In contrast, STA-8666 did not affect tumor features, such as degree of vimentin staining, associated with epithelial-mesenchymal transition (EMT), or downregulate tumor expression of HSP90. These data suggest STA-8666 and other similar targeted compounds may be useful additions to control the growth of early stage NSCLC in patient populations.</p></div