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

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

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

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

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

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

Proteins in A549 cells identified by mass spectrometry whose syntheses are most sensitive to changes in rigidity.

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

Cellular levels of proteins identified by mass spectrometry.

<p><b>A.</b> The levels of actin, tubulin, and phosphofructokinase-1 in A549 cells cultured on soft (150 Pa) or stiff (19200 Pa) gels were analyzed by western blot. Bottom panels show levels of GAPDH as a loading control. <b>B.</b> The levels of actin, tubulin, and PFKP-1 in mPanc96 cells cultured on soft or stiff gels as analyzed by western blot. Bottom panels show levels of GAPDH in cell lysates as a loading control. <b>C.</b> Quantitation of western blot results in (A) and (B). The levels of each protein were normalized to the amount of GAPDH in each sample. The levels of protein in samples prepared from stiff gels were set to 100%, and the levels of protein in the soft samples are displayed as percentage expression of these samples. Results show the mean ± S.E. of at least three experiments. * p<0.05 when compared to the level of actin expression.</p