ZEB1 drives prometastatic actin cytoskeletal remodeling by downregulating miR-34a expression

Metastatic cancer is extremely difficult to treat, and the presence of metastases greatly reduces a cancer patient’s likelihood of long-term survival. The ZEB1 transcriptional repressor promotes metastasis through downregulation of microRNAs (miRs) that are strong inducers of epithelial differentiation and inhibitors of stem cell factors. Given that each miR can target multiple genes with diverse functions, we posited that the prometastatic network controlled by ZEB1 extends beyond these processes. We tested this hypothesis using a mouse model of human lung adenocarcinoma metastasis driven by ZEB1, human lung carcinoma cells, and human breast carcinoma cells. Transcriptional profiling studies revealed that ZEB1 controls the expression of numerous oncogenic and tumor-suppressive miRs, including miR-34a. Ectopic expression of miR-34a decreased tumor cell invasion and metastasis, inhibited the formation of promigratory cytoskeletal structures, suppressed activation of the RHO GTPase family, and regulated a gene expression signature enriched in cytoskeletal functions and predictive of outcome in human lung adenocarcinomas. We identified several miR-34a target genes, including Arhgap1, which encodes a RHO GTPase activating protein that was required for tumor cell invasion. These findings demonstrate that ZEB1 drives prometastatic actin cytoskeletal remodeling by downregulating miR-34a expression and provide a compelling rationale to develop miR-34a as a therapeutic agent in lung cancer patients.Young-Ho Ahn, Don L. Gibbons, Deepavali Chakravarti, Chad J. Creighton, Zain H. Rizvi, Henry P. Adams, Alexander Pertsemlidis, Philip A. Gregory, Josephine A. Wright, Gregory J. Goodall, Elsa R. Flores and Jonathan M. Kuri

Dysregulation of Cell Polarity Proteins Synergize with Oncogenes or the Microenvironment to Induce Invasive Behavior in Epithelial Cells

Changes in expression and localization of proteins that regulate cell and tissue polarity are frequently observed in carcinoma. However, the mechanisms by which changes in cell polarity proteins regulate carcinoma progression are not well understood. Here, we report that loss of polarity protein expression in epithelial cells primes them for cooperation with oncogenes or changes in tissue microenvironment to promote invasive behavior. Activation of ErbB2 in cells lacking the polarity regulators Scribble, Dlg1 or AF-6, induced invasive properties. This cooperation required the ability of ErbB2 to regulate the Par6/aPKC polarity complex. Inhibition of the ErbB2-Par6 pathway was sufficient to block ErbB2-induced invasion suggesting that two polarity hits may be needed for ErbB2 to promote invasion. Interestingly, in the absence of ErbB2 activation, either a combined loss of two polarity proteins, or exposure of cells lacking one polarity protein to cytokines IL-6 or TNFα induced invasive behavior in epithelial cells. We observed the invasive behavior only when cells were plated on a stiff matrix (Matrigel/Collagen-1) and not when plated on a soft matrix (Matrigel alone). Cells lacking two polarity proteins upregulated expression of EGFR and activated Akt. Inhibition of Akt activity blocked the invasive behavior identifying a mechanism by which loss of polarity promotes invasion of epithelial cells. Thus, we demonstrate that loss of polarity proteins confers phenotypic plasticity to epithelial cells such that they display normal behavior under normal culture conditions but display aggressive behavior in response to activation of oncogenes or exposure to cytokines

A Prescription for Excessive Drug Pricing: Leveraging Government Patent Use for Health

High drug prices are creating serious health and fiscal problems in the United States today. This reality is vividly illustrated by recently approved medicines to treat Hepatitis C. These new medicines can cure nearly everyone with this potentially fatal infection and may even enable the elimination of this disease. But the drugs\u27 sticker price- close to $100,000- has meant that very few patients who could benefit from them can access them. This Article describes an approach, available under existing law, to bring about transformative reductions in the prices of these medicines, at least for federal programs and possibly beyond . Under 28 U.S.C. § 1498, the U.S. government can buy generic versions of these medicines at less than 1% of their list price plus a reasonable royalty. This power has received almost no academic attention, despite the fact that it is regularly used by the government in other sectors, including defense. Indeed, though it has now been forgotten, the federal government relied on this provision numerous times to procure cheaper generic drugs in the 1960s. We recover this history and show how § 1498 can once again be used to increase access to life-saving medicines, addressing several important interpretive questions about the application of the provision along the way

Factors associated with successful electrolarynx use after total laryngectomy, a multi‐institutional study

Abstract Objective To identify characteristics associated with successful electrolarynx (EL) use after total laryngectomy (TL). Methods Records of 196 adults who underwent TL from 03/15/2012 to 03/15/2022 at the University of Washington and Puget Sound Veterans Affairs were reviewed. Characteristics included age, Charlson Comorbidity Index, social support, pre‐operative radiation (RT) and chemoradiation (CRT), and 6‐month post‐TL swallow status. EL success was evaluated using pre‐defined criteria of intelligibility, reliability, and independence with use. Poisson regressions and robust standard error estimates were used to estimate unadjusted risk ratios for each characteristic. Statistically significant characteristics were included in multivariate analysis (MVA) to estimate adjusted risk ratios. Results Median age was 64, median Charlson Comorbidity Index was 5, 170 (87%) were male, 159 (81%) had high social support, and 159 (81%) attained post‐TL full‐oral diet. Pre‐operatively, 110 (56%) had RT, including 55 (28%) with CRT. Ninety‐three (47%) met our criteria for EL success. Characteristics significantly associated with EL success included social support (p = .037) and post‐TL full‐oral diet (p = .037); both approached significance on MVA. EL success varied by pre‐operative treatment on univariate (p = .005) and MVA (p = .014). Compared to no prior RT or CRT, the probability of EL success was 29% higher with prior RT and 29% lower with prior CRT in MVA, although these associations did not reach significance. Conclusions In this retrospective review, EL success correlated with high social support, post‐TL full‐oral diet, and pre‐operative treatment history. These results warrant validation in a larger prospective study to help guide the choice of voice rehabilitation modalities or intensified speech therapy. Level of Evidence 4

A Synthetic Matrix with Independently Tunable Biochemistry and Mechanical Properties to Study Epithelial Morphogenesis and EMT in a Lung Adenocarcinoma Model

Better understanding of the biophysical and biochemical cues of the tumor extracellular matrix environment that influence metastasis may have important implications for new cancer therapeutics. Initial exploration into this question has used naturally derived protein matrices that suffer from variability, poor control over matrix biochemistry, and inability to modify the matrix biochemistry and mechanics. Here, we report the use of a synthetic polymer-based scaffold composed primarily of poly(ethylene glycol), or PEG, modified with bioactive peptides to study murine models of lung adenocarcinoma. In this study, we focus on matrix-derived influences on epithelial morphogenesis of a metastatic cell line (344SQ) that harbors mutations in Kras and p53 (trp53) and is prone to a microRNA-200 (miR-200)–dependent epithelial–mesenchymal transition (EMT) and metastasis. The modified PEG hydrogels feature biospecific cell adhesion and cell-mediated proteolytic degradation with independently adjustable matrix stiffness. 344SQ encapsulated in bioactive peptide-modified, matrix metalloproteinase–degradable PEG hydrogels formed lumenized epithelial spheres comparable to that seen with three-dimensional culture in Matrigel. Altering both matrix stiffness and the concentration of cell-adhesive ligand significantly influenced epithelial morphogenesis as manifest by differences in the extent of lumenization, in patterns of intrasphere apoptosis and proliferation, and in expression of epithelial polarity markers. Regardless of matrix composition, exposure to TGF-β induced a loss of epithelial morphologic features, shift in expression of EMT marker genes, and decrease in mir-200 levels consistent with EMT. Our findings help illuminate matrix-derived cues that influence epithelial morphogenesis and highlight the potential utility that this synthetic matrix-mimetic tool has for cancer biology

Combined loss of two regulators from apical and basal polarity complexes is sufficient to induce invasive behavior.

<p>(A) Lysates were immunoblotted to test knockdown for indicated proteins (B) Phase morphology of cells grown on plastic dishes (left panels) or M/Col-1 matrix (right panels). Also refer to <i>SI </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034343#pone.0034343.s002" target="_blank">Fig. S2</a>B. Arrows indicate area of the image magnified in the inset. Scale bars, 100 µm. (C) Quantification of cell invasion plotted as mean ± S.E.M. from at least three independent experiments. *, p<0.005, **, p<0.0001 obtained in an unpaired t-test comparing indicated cell lines with parental MCF10A cells (10A). See Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034343#s4" target="_blank">Methods</a> for details.</p

Loss of one polarity gene and cooperation with pro-tumorigenic cytokines.

<p>(A) Phase images showing induction of invasion in 4-day old acini of parental MCF10A cells or cells expressing shRNAi for indicated polarity genes growing in M/Col-I matrix and treated with 1∶6 diluted supernatant from CpG-treated dendritic cells (DC Sup.), or pro-inflammatory cytokines TNF-α (2 ng/ml) and IL-6 (25 ng/ml) (refer to <i>SI </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034343#pone.0034343.s003" target="_blank">Fig. S3</a>A). Arrows indicate region of the image magnified in the inset. Scale bars, 100 µm (B) Quantification of invasion of DC Sup-treated acini plotted as mean ± S.E.M. from at least three independent experiments. (C–D) Quantification of invasion in MCF10A acini after treatment with recombinant TNF-α (C) or IL-6 (D). Also refer to SI <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034343#pone.0034343.s003" target="_blank">Fig. S3</a>B,C. *, p<0.05, **, p<0.005. p values are based on an unpaired t-test comparing cytokine-treated to the untreated values for the same knockdown.</p

Loss of two polarity genes upregulates EGFR and activate Akt.

<p>(A–B) Parental or knockdown cell lysates were analyzed for EGFR mRNA expression (A) and immunoblotted for EGFR or ErbB2 or ErbB3. Note the increase in EGFR expression without any change in expression of ErbB2 or ErbB3 in polarity knockdown cells. (B) Parental or polarity knockdown cells were grown overnight in low-serum medium without growth factor supplements and next day replenished with Assay medium with 5 ng/ml EGF for indicated times (see Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034343#s4" target="_blank">Methods</a> for details) and immunoblotted for phospho-Akt (Ser-473) first and then stripped and reblotted for pan-Akt. (D) One-day old parental or knockdown MCF10A acini were left untreated or treated with 1nM perifosine and phase images obtained and quantified after 72–96 hr post-perifosine treatment. Data represents mean ± S.E.M. from at least three independent experiments. p<0.05 obtained in an unpaired t-test comparing perifosine-treated to the untreated values for the same knockdown.</p

Loss of Scribble, Dlg1 or AF6 cooperates with ErbB2 activation to promote migration and invasion in MCF-10A cells.

<p>(A) Immunoblot of lysates from 10A.B2 expressing control (Luciferace, 10A.B2.Luc) or Scribble (10A.B2.Scrib) or Dlg1 (10A.B2.Dlg1) or AF6 (10B2.AF6) shRNAs. (B) Transwell cell migration assay in of cell lines in Panel A in the presence (+) or absence (−) of the ErbB2 activator. The graph represents mean of three independent experiments ± S.E.M. *, p<0.05, **, p<0.005. calculated using an unpaired t-test comparing ErbB2-activated polarity-gene knockdown cells with Luc control cells. (Ci-iv) Morphology of 3D acini derived from 10A.B2.Luc,10A.B2.Scrib, 10A.B2.Dlg1 and 10A.B2.AF6 cells grown in M/Col-I in absence (ErbB2−) or presence (ErbB2+) of ErbB2 activator. Scale bars, 100 µm. (Di-ii) M/Col-I grown 10A.B2.Luc or 10A.B2.Scrib acini treated or untreated with ErbB2 activator fixed and immunostained for Laminin (Red) and DAPI-stained for nuclei (Blue). Scale bars, 50 µm. Arrows indicate area of the image magnified in the inset. (E) Percentage of acini showing invasive protrusions were quantified and mean (± S.E.M.) plotted from at least three independent experiments. *, p<0.05 based on an unpaired t-test comparing ErbB2-activated polarity-gene knockdown B2 cells and control Luc.B2 cells. (F) Lysates from 10A.B2 and 10A.B2.Scrib transfected with Par6K19A-Flag (K19 and K19+Scrib) or untransfected (Scrib) cells were immunoblotted for Flag to show Par6.K19A overexpression. ErbB2 blot shows expression levels of ErbB2 in transfected and untransfected lines. (G) Percentage of invasive acini quantified and mean ± S.E.M. plotted for K19, Scrib and K19+Scrib cells. Note the suppression of invasion in K19+Scrib cells compared to Scrib knockdown cells. **, p<0.005 in an unpaired t-test comparing ErbB2− and ErbB2+ in Scrib knockdown cells. See Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034343#s4" target="_blank">Methods</a> for details.</p