Matrix Rigidity Regulates Cancer Cell Growth and Cellular Phenotype

Background: The mechanical properties of the extracellular matrix have an important role in cell growth and differentiation. However, it is unclear as to what extent cancer cells respond to changes in the mechanical properties (rigidity/stiffness) of the microenvironment and how this response varies among cancer cell lines. Methodology/Principal Findings: In this study we used a recently developed 96-well plate system that arrays extracellular matrix-conjugated polyacrylamide gels that increase in stiffness by at least 50-fold across the plate. This plate was used to determine how changes in the rigidity of the extracellular matrix modulate the biological properties of tumor cells. The cell lines tested fall into one of two categories based on their proliferation on substrates of differing stiffness: ‘‘rigidity dependent’ ’ (those which show an increase in cell growth as extracellular rigidity is increased), and ‘‘rigidity independent’’ (those which grow equally on both soft and stiff substrates). Cells which grew poorly on soft gels also showed decreased spreading and migration under these conditions. More importantly, seeding the cell lines into the lungs of nude mice revealed that the ability of cells to grow on soft gels in vitro correlated with their ability to grow in a soft tissue environment in vivo. The lung carcinoma line A549 responded to culture on soft gels by expressing the differentiated epithelial marker E-cadherin and decreasing the expression of the mesenchymal transcription factor Slug. Conclusions/Significance: These observations suggest that the mechanical properties of the matrix environment play

Matrix Rigidity Regulates Cancer Cell Growth by Modulating Cellular Metabolism and Protein Synthesis

Background: Tumor cells in vivo encounter diverse types of microenvironments both at the site of the primary tumor and at sites of distant metastases. Understanding how the various mechanical properties of these microenvironments affect the biology of tumor cells during disease progression is critical in identifying molecular targets for cancer therapy. Methodology/Principal Findings: This study uses flexible polyacrylamide gels as substrates for cell growth in conjunction with a novel proteomic approach to identify the properties of rigidity-dependent cancer cell lines that contribute to their differential growth on soft and rigid substrates. Compared to cells growing on more rigid/stiff substrates (>10,000 Pa), cells on soft substrates (150–300 Pa) exhibited a longer cell cycle, due predominantly to an extension of the G1 phase of the cell cycle, and were metabolically less active, showing decreased levels of intracellular ATP and a marked reduction in protein synthesis. Using stable isotope labeling of amino acids in culture (SILAC) and mass spectrometry, we measured the rates of protein synthesis of over 1200 cellular proteins under growth conditions on soft and rigid/stiff substrates. We identified cellular proteins whose syntheses were either preferentially inhibited or preserved on soft matrices. The former category included proteins that regulate cytoskeletal structures (e.g., tubulins) and glycolysis (e.g., phosphofructokinase-1), whereas the latter category included proteins that regulate key metabolic pathways required for survival, e.g., nicotinamide phosphoribosyltransferase, a regulator of the NAD salvage pathway. Conclusions/Significance: The cellular properties of rigidity-dependent cancer cells growing on soft matrices are reminiscent of the properties of dormant cancer cells, e.g., slow growth rate and reduced metabolism. We suggest that the use of relatively soft gels as cell culture substrates would allow molecular pathways to be studied under conditions that reflect the different mechanical environments encountered by cancer cells upon metastasis to distant sites

The differential effects of ecstasy/polydrug use on executive components: shifting, inhibition, updating and access to semantic memory

Rationale/Objectives Recent theoretical models suggest that the central executive may not be a unified structure. The present study explored the nature of central executive deficits in ecstasy users. Methods In study 1, 27 ecstasy users and 34 non-users were assessed using tasks to tap memory updating (computation span; letter updating) and access to long-term memory (a semantic fluency test and the Chicago Word Fluency Test). In study 2, 51 ecstasy users and 42 non-users completed tasks that assess mental set switching (number/letter and plus/minus) and inhibition (random letter generation). Results MANOVA revealed that ecstasy users performed worse on both tasks used to assess memory updating and on tasks to assess access to long-term memory (C- and S-letter fluency). However, notwithstanding the significant ecstasy group-related effects, indices of cocaine and cannabis use were also significantly correlated with most of the executive measures. Unexpectedly, in study 2, ecstasy users performed significantly better on the inhibition task, producing more letters than non-users. No group differences were observed on the switching tasks. Correlations between indices of ecstasy use and number of letters produced were significant. Conclusions The present study provides further support for ecstasy/polydrug-related deficits in memory updating and in access to long-term memory. The surplus evident on the inhibition task should be treated with some caution, as this was limited to a single measure and has not been supported by our previous work

Inhibition of the Growth of Patient-Derived Pancreatic Cancer Xenografts with the MEK Inhibitor Trametinib Is Augmented by Combined Treatment with the Epidermal Growth Factor Receptor/HER2 Inhibitor Lapatinib

Mutations of the oncogene KRAS are important drivers of pancreatic cancer progression. Activation of epidermal growth factor receptor (EGFR) and human EGFR2 (HER2) is observed frequent in pancreatic adenocarcinomas. Because of co-activation of these two signaling pathways, we assessed the efficacy of inhibition of EGFR/HER2 receptors and the downstream KRAS effector, mitogen-activated protein kinase/extracellular-signal regulated kinase (ERK) kinase 1 and 2 (MEK1/2), on pancreatic cancer proliferation in vitro and in a murine orthotopic xenograft model. Treatment of established and patient-derived pancreatic cancer cell lines with the MEK1/2 inhibitor trametinib (GSK1120212) inhibited proliferation, and addition of the EGFR/HER2 inhibitor lapatinib enhanced the inhibition elicited by trametinib in three of eight cell lines. Importantly, in the orthotopic xenograft model, treatment with lapatinib and trametinib resulted in significantly enhanced inhibition of tumor growth relative to trametinib treatment alone in four of five patient-derived tumors tested and was, in all cases, significantly more effective in reducing the size of established tumors than treatment with lapatinib or trametinib alone. Acute treatment of established tumors with trametinib resulted in an increase in AKT2 phosphorylation that was blunted in mice treated with both trametinib and lapatinib. These data indicate that inhibition of the EGFR family receptor signaling may contribute to the effectiveness of MEK1/2 inhibition of tumor growth possibly through the inhibition of feedback activation of receptor tyrosine kinases in response to inhibition of the RAS-RAF-MEK-ERK pathway. These studies provide a rationale for assessing the co-inhibition of these pathways in the treatment of pancreatic cancer patients

BrdU pulse-chase of cell cycle progression.

<p>A549 cells were pulsed with BrdU for 30 minutes following growth on soft or stiff gels for 2 days. <b>A.</b> Labeling cells with BrDU for cell cycle analysis. Cells are pulsed with BrDU for 30 min, and the BrDU-positive population is followed over time as it transitions through the phases of the cell cycle. <b>B.</b> Scatter plot histograms of BrdU-labeled cells on soft (top panel) or stiff (bottom panel) gels, stained for DNA content (X-axis) and BrdU (Y-axis). The times indicated are the times, in hours, after the BrdU pulse. <b>C.</b> Cell cycle progression analysis was performed on the scatter plot histograms from the cells grown on gels for 2 days (left) or 5 days (right).</p

Identification of proteins that are differentially regulated by rigidity.

<p>H/L ratios of proteins identified in both stiff and soft samples plotted against each other for A549 cells (left) and mPanc96 cells (right). H/L ratios from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037231#pone-0037231-g004" target="_blank">Figure 4</a> were quantile normalized and t-tests were performed using estimated variability (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037231#s4" target="_blank">Methods</a>) to identify individual proteins with relatively different synthesis rates between stiff and soft samples. Proteins that are synthesized faster (relatively lower H/L ratio and p-value<0.05) in soft samples (compared to stiff samples) are shown in red, and proteins that are synthesized slower in soft samples are shown in green.</p

Protein synthesis is decreased in rigidity-dependent cells cultured on soft gels.

<p>A549 cells were subjected to SILAC analysis to determine rates of protein synthesis on soft or stiff gels. <b>A.</b> Overview of the SILAC procedure. A549 cells were cultured on soft or stiff gels for 4 days in the presence of “heavy” media, followed by a 24-hour incubation with “light” media. The cells were lysed, cellular proteins were digested with trypsin, and the resulting peptides were analyzed by mass spectrometry. <b>B.</b> Boxplots of heavy to light (H/L) ratios of proteins from A549 cells (left) or mPanc96 cells (right) grown on stiff (19200 Pa) or soft (150 Pa) substrates. H/L ratio distributions are significantly different between stiff and soft for A549 cells but not for mPanc96 cells using two-tailed unpaired t-tests. The boxes contain the data between the 25 and 75 percentile, and the line within the box indicates the median. The dashed line at the top of the graph marks the upper limit, above which the outliers were truncated.</p

Cyclin D1 expression in rigidity-dependent cells growing on soft and stiff gels.

<p>A549 cells and MDA-MB-231 cells were cultured on 150 Pa, 4800 Pa, or 19200 Pa polyacrylamide gels for 2 or 5 days. Cells were lysed and analyzed by western blot for the expression of cyclin D1 (top panel). The expression of GAPDH was analyzed as a loading control (bottom panel). The blot is representative of three experiments.</p

Proteins in mPanc96 cells identified by mass spectrometry whose rates of syntheses decrease when cultured on soft gels.

<p>The table lists proteins that exhibited higher heavy/light ratios (and therefore, lower synthesis) on soft gels compared to the mean.</p