MAP2K4 increases HSP27 and MMP-2 expression <i>in vivo</i>.

<p><b>A</b>) MMP transcript expression of tumor samples. The levels of MMP-2, MMP-9, MMP-10 and MMP-13 transcript levels were measured by qRT/PCR, normalized to GAPDH, and expressed as the mean ± SEM percentage of VC. Each tumor sample was run twice, in duplicates of N = 2, and compared to a reference sample. <b>B-F</b>) Quantification of phospho- and total protein expression in tumor samples. Protein expression was measured by Western Blot, twice for each tumor, compared to a reference sample, and graphed data are the mean ± SEM percent of VC. * denotes p≤0.05 between the indicated groups.</p

MAP2K4 increases cellular invasion, MMP-2, and HSP27 in early-stage cancer cell lines.

<p><b>A</b>) MAP2K4 protein expression in LNCaP and 1542CPTX cell lines by Western Blot. <b>B</b>) Relative cellular invasion of LNCaP and 1542CPTX (42C) cell lines as measured by a Matrigel coated Boyden chamber. Data are from four independent experiments, each in replicates of N = 2. * denotes p≤0.05 between the indicated groups. <b>C</b>) Relative MMP-2 mRNA expression in LNCaP and 42C cell lines as measured by qRT/PCR. Data are from five independent experiments, each in replicates of N = 2. * denotes p≤0.05 between the indicated groups. <b>D</b>) Phosphorylated and total HSP27 expression in LNCaP and 1542CPTX cell lines by Western Blot.</p

MAP2K4 Overexpression Promotes Human Prostate Cancer Cell Invasion and Metastasis.

<p><b>A</b>) MAP2K4 protein expression in MAP2K4 variant cell lines. The expression of MAP2K4 protein was measured in wild type, WT1, WT2, and constitutive active, CA1, CA2, CA3, MAP2K4 over expressing cell lines, and vector control, VC1, VC2, cell lines by Western blot. A representative Western blot is depicted, with quantification of the MAP2K4/GAPDH levels, normalized to VC1 cells, shown above each lane. <b>B</b>) Invasion of MAP2K4 variant cell lines. Boyden chamber Matrigel cell invasion assays were performed, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102289#s2" target="_blank">Methods</a>. Data are from 5 independent experiments, each in replicates of N = 3. Data from the respective wild type, constitutive active and vector control cell lines were combined to give an average read out for WT-MAP2K4, CA-MAP2K4 and VC cells, respectively. Data are expressed as the mean ± SEM percentage of VC cells. <b>C-E</b>) The effect of MAP2K4 expression upon metastatic spread and tumor growth. Equal numbers of mice were orthotopically implanted with VC1 or VC2 cells (forming the VC cohort), WT1 or WT2 cells (WT cohort), or with CA1, CA2 or CA3 cells (CA cohort). In (C) the resultant number of distant metastasis is expressed as the mean ± SEM percentage of VC mice, in (E) the number of mice developing circulating tumor cells is depicted, and in (F) tumor volume is graphed as the mean ± SEM percentage of VC mice. A representative image of a distinct metastasis is shown in (D). * denotes p≤0.05 between VC and an experimental group.</p

MAP2K4 overexpression specifically alters HSP27 phosphorylation and total protein expression.

<p>The expression of total and phosphorylated forms of HSP27 proteins were assessed by Western blot in the indicated cell lines, <b>A-B</b>. Data from three separate blots are graphically depicted in<b>B</b>, as mean ± SEM. <b>C</b>), HSP27 transcript levels were measured by qRT/PCR, normalized to GAPDH, and expressed as the mean ± SEM percentage of VC. Data are from three independent experiments, each in replicates of N = 2. * denotes p≤0.05 between the indicated groups.</p

MAP2K4 alters protease production, but not cell migration or cell growth <i>in vitro</i>.

<p>Studies were conducted on MAP2K4 variant cell lines. <b>A</b>) Knockdown of MAP2K4 protein by siRNA. After transfection of cells with siRNA targeting MAP2K4 (siMAP2K4) or non-targeting control (siCON), MAP2K4 expression was measured by Western blot. Percent knockdown is denoted above the blot. <b>B</b>) Effect of MAP2K4 knockdown on cell invasion. MAP2K4 variant cell lines were treated with siRNA, as indicated, and cell invasion measured and depicted as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102289#pone-0102289-g001" target="_blank">Fig 1</a>. Data are from four experiments, each in replicates of N = 2, and are expressed as the percentage of invading cells. <b>C</b>) Effects on cell migration. Cell migration was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102289#s2" target="_blank">Methods</a>. Data from individual types of clones were combined, are from three independent experiments, each in replicates of N = 3, and are expressed as the percentage of migrating cells, normalized to VC. <b>D</b>) MMP transcript expression. The levels of MMP-2, MMP-9, MMP-10 and MMP-13 transcript levels were measured by qRT/PCR, normalized to GAPDH, and expressed as the percentage of VC. Data are from four independent experiments, each in replicates of N = 2. <b>E</b>) Cell growth. Cells were plated at the indicated concentrations, allowed to grow for 5 days, MTT added, and optical density determined. Data are from three independent experiments, each N = 3. <b>F</b>) Colony formation. Colony formation after 14 days was determined in three independent experiments, each N = 2, and expressed as the percent of VC. Values in all graphs are the mean ± SEM. * denotes p≤0.05 between the indicated groups.</p

MAP2K4's pro-invasive effects are dependent on HSP27 and MMP-2.

<p><b>A</b>) Knockdown of HSP27 protein by siRNA. After transfection of cells with siRNA targeting HSP27 (siHSP27) or non-targeting control (siCON), HSP27 expression was measured by Western blot. <b>B</b>) Effect of HSP27 knockdown on cell invasion. MAP2K4 variant cell lines were treated with siRNA, as indicated, and cell invasion measured and depicted as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102289#pone-0102289-g001" target="_blank">Fig 1</a>. Data are from four experiments, each in replicates of N = 2, and are expressed as the percentage of invading cells. <b>C</b>) Knockdown of MMP-2 mRNA by siRNA. After transfection of cells with siRNA targeting MMP-2 (siMMP2) or non-targeting control (siCON), MMP-2 expression was measured by qRT/PCR. <b>D</b>) Effect of MMP-2 knockdown on cell invasion. MAP2K4 variant cell lines were treated with siRNA, as indicated, and cell invasion measured and depicted as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102289#pone-0102289-g001" target="_blank">Fig 1</a>. Data are from four experiments, each in replicates of N = 2, and are expressed as the percentage of invading cells.</p

Prevention of colonic neoplasia with polyethylene glycol: A short term randomized placebo-controlled double-blinded trial

<div><p>Chemoprevention represents an attractive modality against colorectal cancer (CRC) although widespread clinical implementation of promising agents (e.g. aspirin/NSAIDS) have been stymied by both suboptimal efficacy and concerns over toxicity. This highlights the need for better agents. Several groups, including our own, have reported that the over-the-counter laxative polyethylene glycol (PEG) has remarkable efficacy in rodent models of colon carcinogenesis. In this study, we undertook the first randomized human trial to address the role of PEG in prevention of human colonic neoplasia. This was a double-blind, placebo-controlled, three-arm trial where eligible subjects were randomized to 8g PEG-3350 (n = 27) or 17g PEG-3350 (n = 24), or placebo (n = 24; maltodextrin) orally for a duration of six months. Our initial primary endpoint was rectal aberrant crypt foci (ACF) but this was changed during protocol period to rectal mucosal epidermal growth factor receptor (EGFR). Of the 87 patients randomized, 48 completed study primary endpoints and rectal EGFR unchanged PEG treatment. Rectal ACF had a trend suggesting potentially reduction with PEG treatment (pre-post change 1.7 in placebo versus -0.3 in PEG 8+ 17g doses, p = 0.108). Other endpoints (proliferation, apoptosis, expression of SNAIL and E-cadherin), previously noted to be modulated in rodent models, appeared unchanged with PEG treatment in this clinical trial. We conclude that PEG was generally well tolerated with the trial failing to meet primary efficacy endpoints. However, rectal ACFs demonstrated a trend (albeit statistically insignificant) for suppression with PEG. Moreover, all molecular assays including EGFR were unaltered with PEG underscoring issues with lack of translatability of biomarkers from preclinical to clinical trials. This data may provide the impetus for future clinical trials on PEG using more robust biomarkers of chemoprevention.</p><p><b>Trial registration:</b> ClinicalTrials.gov <a href="https://clinicaltrials.gov/ct2/show/NCT00828984" target="_blank">NCT00828984</a></p></div

Prevention of colonic neoplasia with polyethylene glycol: A short term randomized placebo-controlled double-blinded trial

<div><p>Chemoprevention represents an attractive modality against colorectal cancer (CRC) although widespread clinical implementation of promising agents (e.g. aspirin/NSAIDS) have been stymied by both suboptimal efficacy and concerns over toxicity. This highlights the need for better agents. Several groups, including our own, have reported that the over-the-counter laxative polyethylene glycol (PEG) has remarkable efficacy in rodent models of colon carcinogenesis. In this study, we undertook the first randomized human trial to address the role of PEG in prevention of human colonic neoplasia. This was a double-blind, placebo-controlled, three-arm trial where eligible subjects were randomized to 8g PEG-3350 (n = 27) or 17g PEG-3350 (n = 24), or placebo (n = 24; maltodextrin) orally for a duration of six months. Our initial primary endpoint was rectal aberrant crypt foci (ACF) but this was changed during protocol period to rectal mucosal epidermal growth factor receptor (EGFR). Of the 87 patients randomized, 48 completed study primary endpoints and rectal EGFR unchanged PEG treatment. Rectal ACF had a trend suggesting potentially reduction with PEG treatment (pre-post change 1.7 in placebo versus -0.3 in PEG 8+ 17g doses, p = 0.108). Other endpoints (proliferation, apoptosis, expression of SNAIL and E-cadherin), previously noted to be modulated in rodent models, appeared unchanged with PEG treatment in this clinical trial. We conclude that PEG was generally well tolerated with the trial failing to meet primary efficacy endpoints. However, rectal ACFs demonstrated a trend (albeit statistically insignificant) for suppression with PEG. Moreover, all molecular assays including EGFR were unaltered with PEG underscoring issues with lack of translatability of biomarkers from preclinical to clinical trials. This data may provide the impetus for future clinical trials on PEG using more robust biomarkers of chemoprevention.</p><p><b>Trial registration:</b> ClinicalTrials.gov <a href="https://clinicaltrials.gov/ct2/show/NCT00828984" target="_blank">NCT00828984</a></p></div

Changes in mean values of biomarkers in treated and placebo groups, with high and low PEG doses combined.

<p>Changes in mean values of biomarkers in treated and placebo groups, with high and low PEG doses combined.</p

Demographics of study subjects.

<p>Demographics of study subjects.</p