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

PAK1 phosphorylation of MEK1 regulates fibronectin-stimulated MAPK activation

Activation of the Ras–MAPK signal transduction pathway is necessary for biological responses both to growth factors and ECM. Here, we provide evidence that phosphorylation of S298 of MAPK kinase 1 (MEK1) by p21-activated kinase (PAK) is a site of convergence for integrin and growth factor signaling. We find that adhesion to fibronectin induces PAK1-dependent phosphorylation of MEK1 on S298 and that this phosphorylation is necessary for efficient activation of MEK1 and subsequent MAPK activation. The rapid and efficient activation of MEK and phosphorylation on S298 induced by cell adhesion to fibronectin is influenced by FAK and Src signaling and is paralleled by localization of phospho-S298 MEK1 and phospho-MAPK staining in peripheral membrane–proximal adhesion structures. We propose that FAK/Src-dependent, PAK1-mediated phosphorylation of MEK1 on S298 is central to the organization and localization of active Raf–MEK1–MAPK signaling complexes, and that formation of such complexes contributes to the adhesion dependence of growth factor signaling to MAPK

Decreased Peritoneal Ovarian Cancer Growth in Mice Lacking Expression of Lipid Phosphate Phosphohydrolase 1

<div><p>Lysophosphatidic acid (LPA) is a bioactive lipid that enhances ovarian cancer cell proliferation, migration and invasion <i>in vitro</i> and stimulates peritoneal metastasis <i>in vivo</i>. LPA is generated through the action of autotaxin or phospholipases, and degradation begins with lipid phosphate phosphohydrolase (LPP)-dependent removal of the phosphate. While the effects of LPA on ovarian cancer progression are clear, the effects of LPA metabolism within the tumor microenvironment on peritoneal metastasis have not been reported. We examined the contribution of lipid phosphatase activity to ovarian cancer peritoneal metastasis using mice deficient in LPP1 expression. Homozygous deletion of LPP1 (LPP1 KO) results in elevated levels and decreased turnover of LPA <i>in vivo</i>. Within 2 weeks of intraperitoneal injection of syngeneic mouse ovarian cancer cells, we observed enhanced tumor seeding in the LPP1 KO mice compared to wild type. However, tumor growth plateaued in the LPP1 KO mice by 3 weeks while tumors continued to grow in wild type mice. The decreased tumor burden was accompanied by increased apoptosis and no change in proliferation or angiogenesis. Tumor growth was restored and apoptosis reversed with exogenous administration of LPA. Together, these observations demonstrate that the elevated levels of LPA per se in LPP1 KO mice do not inhibit tumor growth. Rather, the data support the notion that either elevated LPA concentration or altered LPA metabolism affects other growth-promoting contributions of the tumor microenvironment.</p></div

Increased peritoneal tumor seeding in LPP1 KO mice.

<p>ID8ip2Luc ovarian cancer cells were injected IP into C57BL/6 (WT) or LPP1 KO mice. <b>(A)</b> Mice (WT n = 10; LPP1 KO n = 12) were imaged weekly following tumor initiation to monitor tumor growth. Data represent mean total flux (photons/second) ± std err and were analyzed by 2-way ANOVA followed by Tukey’s multiple comparisons test. <b>(B)</b> The total number of microscopic tumor nodules was counted in an individual, randomly selected, H&E stained section of omentum per mouse obtained 2 weeks after tumor initiation (WT n = 7; LPP1 KO n = 8). Data represent mean ± std err; p = 0.0069 determined by Student’s t-test. <b>(C)</b> The percentage of wild type or LPP1 KO (from <b>(B)</b>) mice with invasive (solid bars) or non-invasive (includes no tumor; stippled bars) tumors 2 weeks after tumor initiation. Number of mice per outcome is indicated. Significance determined by Fisher’s Exact Test. <b>(D)</b> The percentage wild type or LPP1 KO (from <b>(B)</b>) mice with (positive, solid bars) or without (negative, stippled bars) mesothelial VCAM-1 staining by IHC 2 weeks after tumor initiation. Number of mice per outcome is indicated. Significance determined by Fisher’s Exact Test.</p

Angiogenesis is not defective in LPP1 KO mice.

<p><b>(A)</b> Tumors from mice 8 weeks after initiation were sectioned and stained for CD31, and the total number of CD31 positive vessels per tumor area from 5 high powered fields (hpf; 200X magnification) per mouse for wild type (WT, n = 5) and LPP1 KO (n = 5) mice is plotted. The total number of CD31 positive vessels observed in 5 hpf of matrigel plugs containing conditioned media from ID8ip2Luc cells <b>(B)</b> or FGF/VEGF <b>(C)</b> is plotted (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120071#sec002" target="_blank">Materials and Methods</a>). Data are presented with Box and Whiskers plots as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120071#pone.0120071.g003" target="_blank">Fig. 3</a>. *p = 0.0083, Student’s t-test.</p

Daily injection of LPA rescues tumor suppression and stimulates ovarian cancer growth.

<p>ID8ip2Luc ovarian cancer cells were injected IP into wild type (WT, n = 19) or LPP1 KO (n = 20) mice. Mice received daily injections of LPA (n = 6 WT, n = 7 LPP1 KO) or vehicle (n = 13 for both genotypes) beginning the day after tumor injection. <b>(A)</b> Mice were imaged weekly following tumor initiation to monitor tumor growth. Data represent mean total flux (photons/second) ± std err; *p < 0.001, 2-way ANOVA followed by Tukey’s multiple comparisons test. Tumors were collected 6 weeks after initiation, sectioned and stained for Ki67 <b>(B)</b> or cleaved caspase 3 <b>(C).</b> Quantification of Ki67 and cleaved caspase 3 was achieved by determining the percentage of positively stained cells in 5 high-powered fields (400x magnification) per mouse, 5 mice per group. Data are presented with Box and Whiskers plots as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120071#pone.0120071.g003" target="_blank">Fig. 3</a>. **p < 0.0001, 2-way ANOVA followed by Tukey’s multiple comparisons test.</p

Mitogen-Activated Protein Kinase Feedback Phosphorylation Regulates MEK1 Complex Formation and Activation during Cellular Adhesion

Cell adhesion and spreading depend on activation of mitogen-activated kinase, which in turn is regulated both by growth factor and integrin signaling. Growth factors, such as epidermal growth factor, are capable of activating Ras and Raf, but integrin signaling is required to couple Raf to MEK and MEK to extracellular signal-regulated protein kinase (ERK). It was previously shown that Rac-p21-activated kinase (PAK) signaling regulated the physical association of MEK1 with ERK2 through phosphorylation sites in the proline-rich sequence (PRS) of MEK1. It was also shown that activation of MEK1 and ERK by integrins depends on PAK phosphorylation of S298 in the PRS. Here we report a novel MEK1-specific regulatory feedback mechanism that provides a means by which activated ERK can terminate continued PAK phosphorylation of MEK1. Activated ERK can phosphorylate T292 in the PRS, and this blocks the ability of PAK to phosphorylate S298 and of Rac-PAK signaling to enhance MEK1-ERK complex formation. Preventing ERK feedback phosphorylation on T292 during cellular adhesion prolonged phosphorylation of S298 by PAK and phosphorylation of S218 and S222, the MEK1 activating sites. We propose that activation of ERK during adhesion creates a feedback system in which ERK phosphorylates MEK1 on T292, and this in turn blocks additional S298 phosphorylation in response to integrin signaling

Notch3 signaling promotes tumor cell adhesion and progression in a murine epithelial ovarian cancer model.

High grade serous ovarian cancer (HGSC) is the most common and deadly type of ovarian cancer, largely due to difficulties in early diagnosis and rapid metastasis throughout the peritoneal cavity. Previous studies have shown that expression of Notch3 correlates with worse prognosis and increased tumorigenic cell behaviors in HGSC. We investigated the mechanistic role of Notch3 in a model of metastatic ovarian cancer using the murine ovarian surface epithelial cell line, ID8 IP2. Notch3 was activated in ID8 IP2 cells via expression of the Notch3 intracellular domain (Notch3IC). Notch3IC ID8 IP2 cells injected intraperitoneally caused accelerated ascites and reduced survival compared to control ID8 IP2, particularly in early stages of disease. We interrogated downstream targets of Notch3IC in ID8 IP2 cells by RNA sequencing and found significant induction of genes that encode adhesion and extracellular matrix proteins. Notch3IC ID8 IP2 showed increased expression of ITGA1 mRNA and cell-surface protein. Notch3IC-mediated increase of ITGA1 was also seen in two human ovarian cancer cells. Notch3IC ID8 IP2 cells showed increased adhesion to collagens I and IV in vitro. We propose that Notch3 activation in ovarian cancer cells causes increased adherence to collagen-rich peritoneal surfaces. Thus, the correlation between increased Notch3 signaling and poor prognosis may be influenced by increased metastasis of HGSC via increased adherence of disseminating cells to new metastatic sites in the peritoneum