Inhibition of cancer cell invasion and metastasis by genistein

Genistein is a small, biologically active flavonoid that is found in high amounts in soy. This important compound possesses a wide variety of biological activities, but it is best known for its ability to inhibit cancer progression. In particular, genistein has emerged as an important inhibitor of cancer metastasis. Consumption of genistein in the diet has been linked to decreased rates of metastatic cancer in a number of population-based studies. Extensive investigations have been performed to determine the molecular mechanisms underlying genistein’s antimetastatic activity, with results indicating that this small molecule has significant inhibitory activity at nearly every step of the metastatic cascade. Reports have demonstrated that, at high concentrations, genistein can inhibit several proteins involved with primary tumor growth and apoptosis, including the cyclin class of cell cycle regulators and the Akt family of proteins. At lower concentrations that are similar to those achieved through dietary consumption, genistein can inhibit the prometastatic processes of cancer cell detachment, migration, and invasion through a variety of mechanisms, including the transforming growth factor (TGF)-β signaling pathway. Several in vitro findings have been corroborated in both in vivo animal studies and in early-phase human clinical trials, demonstrating that genistein can both inhibit human cancer metastasis and also modulate markers of metastatic potential in humans, respectively. Herein, we discuss the variety of mechanisms by which genistein regulates individual steps of the metastatic cascade and highlight the potential of this natural product as a promising therapeutic inhibitor of metastasis

The genetic architecture of the human cerebral cortex

The cerebral cortex underlies our complex cognitive capabilities, yet little is known about the specific genetic loci that influence human cortical structure. To identify genetic variants that affect cortical structure, we conducted a genome-wide association meta-analysis of brain magnetic resonance imaging data from 51,665 individuals. We analyzed the surface area and average thickness of the whole cortex and 34 regions with known functional specializations. We identified 199 significant loci and found significant enrichment for loci influencing total surface area within regulatory elements that are active during prenatal cortical development, supporting the radial unit hypothesis. Loci that affect regional surface area cluster near genes in Wnt signaling pathways, which influence progenitor expansion and areal identity. Variation in cortical structure is genetically correlated with cognitive function, Parkinson's disease, insomnia, depression, neuroticism, and attention deficit hyperactivity disorder

Mitogen-activated protein kinase kinase 4 (MAP2K4) promotes human prostate cancer metastasis.

Prostate cancer (PCa) is the second leading cause of cancer death in the US. Death from PCa primarily results from metastasis. Mitogen-activated protein kinase kinase 4 (MAP2K4) is overexpressed in invasive PCa lesions in humans, and can be inhibited by small molecule therapeutics that demonstrate favorable activity in phase II studies. However, MAP2K4's role in regulating metastatic behavior is controversial and unknown. To investigate, we engineered human PCa cell lines which overexpress either wild type or constitutive active MAP2K4. Orthotopic implantation into mice demonstrated MAP2K4 increases formation of distant metastasis. Constitutive active MAP2K4, though not wild type, increases tumor size and circulating tumor cells in the blood and bone marrow. Complementary in vitro studies establish stable MAP2K4 overexpression promotes cell invasion, but does not affect cell growth or migration. MAP2K4 overexpression increases the expression of heat shock protein 27 (HSP27) protein and protease production, with the largest effect upon matrix metalloproteinase 2 (MMP-2), both in vitro and in mouse tumor samples. Further, MAP2K4-mediated increases in cell invasion are dependent upon heat shock protein 27 (HSP27) and MMP-2, but not upon MAP2K4's immediate downstream targets, p38 MAPK or JNK. We demonstrate that MAP2K4 increases human PCa metastasis, and prolonged over expression induces long term changes in cell signaling pathways leading to independence from p38 MAPK and JNK. These findings provide a mechanistic explanation for human studies linking increases in HSP27 and MMP-2 to progression to metastatic disease. MAP2K4 is validated as an important therapeutic target for inhibiting human PCa metastasis

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 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'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

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