Epac1‐induced cellular proliferation in prostate cancer cells is mediated by B‐Raf/ERK and mTOR signaling cascades

cAMP‐dependent, PKA‐independent effects on cell proliferation are mediated by cAMP binding to EPAC and activation of Rap signaling. In this report, we employed the analogue 8‐CPT‐2‐O‐Me‐cAMP to study binding to EPAC and subsequent activation of B‐Raf/ERK and mTOR signaling in human cancer cells. This compound significantly stimulated DNA synthesis, protein synthesis, and cellular proliferation of human 1‐LN prostate cancer cells. By study of phosphorylation‐dependent activation, we demonstrate that EPAC‐mediated cellular effects require activation of the B‐Raf/ERK and mTOR signaling cascades. RNAi directed against EPAC gene expression as well as inhibitors of ERK, PI 3‐kinase, and mTOR were employed to further demonstrate the role of these pathways in regulating prostate cancer cell proliferation. These studies were then extended to several other human prostate cancer cell lines and melanoma cells with comparable results. We conclude that B‐Raf/ERK and mTOR signaling play an essential role in cAMP‐dependent, but PKA‐independent, proliferation of cancer cells. J. Cell. Biochem. 108: 998–1011, 2009. © 2009 Wiley‐Liss, Inc.     

Opposing effects of protein kinase Calpha and protein kinase Cepsilon on collagen expression by human lung fibroblasts are mediated via MEK/ERK and caveolin-1 signaling

The roles of MEK, ERK, the epsilon and alpha isoforms of protein kinase C (PKC), and caveolin-1 in regulating collagen expression were studied in normal lung fibroblasts. Knocking down caveolin-1 gave particularly striking results. A 70% decrease caused a 5-fold increase in MEK/ERK activation and collagen expression. The combined data reveal a branched signaling pathway. In its central portion MEK activates ERK, leading to increased collagen expression. Two branches converge on MEK/ERK. In one, increased PKCepsilon leads to MEK/ERK activation. In another, increased PKCalpha induces caveolin-1 expression, which in turn inhibits MEK/ERK activation and collagen expression. Lung fibroblasts from scleroderma patients with pulmonary fibrosis showed altered signaling. Consistent with their overexpression of collagen, scleroderma lung fibroblasts contain more activated MEK/ERK and less caveolin-1 than normal lung fibroblasts. Because cutaneous fibrosis is the hallmark of scleroderma, we also studied dermal fibroblasts. As in lung, there was more activated MEK/ERK in cells from scleroderma patients than in control cells, and MEK inhibition decreased collagen expression. However, the distinctive levels of PKCepsilon, PKCalpha, and caveolin-1 in lung and dermal fibroblasts from scleroderma patients and control subjects indicate that the links between these signaling proteins and MEK/ERK must function differently in the four cell types. Finally, we confirmed the relevance of these signaling cascades in vivo. The combined results demonstrate that a branched signaling pathway involving MEK, ERK, PKCepsilon, PKCalpha, and caveolin-1 regulates collagen expression in normal lung tissue and is perturbed during fibrosis.     

Extracellular vesicles from malignant effusions induce tumor cell migration: inhibitory effect of LMWH tinzaparin

Elevated levels of extracellular vesicles (EVs) have been correlated with inflammatory diseases as well as progressive and metastatic cancer. By presenting tissue factor (TF) on their membrane surface, cellular microparticles (MPs) activate both the coagulation system and cell‐signaling pathways such as the PAR/ERK pathway. We have shown before that malignant effusions are a rich source of tumor cell‐derived EVs. Here, we used EVs from malignant effusions from three different patients after serial low‐speed centrifugation steps as recommended by the ISTH (lsEV). Significant migration of human pancreatic carcinoma cells could be induced by lsEVs and was effectively inhibited by pre‐incubation with tinzaparin, a low‐molecular‐weight heparin. Tinzaparin induced tissue factor pathway inhibitor (TFPI) release from tumor cells, and recombinant TFPI inhibited EV‐induced tumor cell migration. EVs also induced ERK phosphorylation, whereas inhibitors of PAR2 and ERK suppressed EV‐induced tumor cell migration. LsEVs have been characterized by high‐resolution flow cytometry and, after elimination of smaller vesicles including exosomes, by further high‐speed centrifugation (hsEV). The remaining population consisting primarily of MPs is indeed the main migration‐inducing population with tenase activity. Compared to other LMWHs, tinzaparin is suggested to have high potency to induce TFPI release from epithelial cells. The migration‐inhibitory effect of TFPI and the interruption of tumor cell migration by inhibitors of PAR2 and ERK suggest that lsEVs induce tumor cell migration by activating the PAR2 signaling pathway. Tinzaparin might inhibit this process at least partly by inducing the release of TFPI from tumor cells, which blocks PAR‐activating TF complexes. The clinical relevance of the results is discussed.     

km23‐1/DYNLRB1 regulation of MEK/ERK signaling and R‐Ras in invasive human colorectal cancer cells

We previously found that km23‐1/DYNLRB1 is required for transforming growth factor‐β (TGFβ) production through Ras/ERK pathways in TGFβ‐sensitive epithelial cells and in human colorectal cancer (CRC) cells. Here we demonstrate that km23‐1/DYNLRB1 is required for mitogen‐activated protein kinase kinase (MEK) activation in human CRC cells, detected by km23‐1/DYNLRB1‐siRNA inhibition of phospho‐(p)‐MEK immunostaining in RKO cells. Furthermore, we show that CRISPR‐Cas9 knock‐out (KO) of km23‐1/DYNLRB1 reduced cell migration in two additional CRC models, HCT116 and DLD‐1. Of interest, in contrast to our previous work showing that dynein motor activity was required for TGFβ‐mediated nuclear translocation of Smad2, in the current report, we demonstrate for the first time that disruption of dynein motor activity did not reduce TGFβ‐mediated activation of MEK1/2 or c‐Jun N‐terminal kinase (JNK). Moreover, size exclusion chromatography of RKO cell lysates revealed that B‐Raf, extracellular signal‐regulated kinase (ERK), and p‐ERK were not present in the large molecular weight fractions containing dynein holocomplex components. Furthermore, sucrose gradient fractionation of cell lysates from both HCT116 and CBS CRC cells demonstrated that km23‐1/DYNLRB1 co‐sedimented with Ras, p‐ERK, and ERK in fractions that did not contain components of holo‐dynein. Thus, km23‐1/DYNLRB1 may be associated with activated Ras/ERK signaling complexes in cell compartments that do not contain the dynein holoprotein complex, suggesting dynein‐independent km23‐1/DYNLRB1 functions in Ras/ERK signaling. Finally, of the Ras isoforms, R‐Ras is most often associated with cell migration, adhesion, and protrusive activity. Here, we show that a significant fraction of km23‐1/DYNLRB1 and RRas wase co‐localized at the protruding edges of migrating HCT116 cells, suggesting an important role for the km23‐1/DYNLRB1‐R‐Ras complex in CRC invasion.     

Cell‐specific responses to the cytokine TGFβ are determined by variability in protein levels

The cytokine TGFβ provides important information during embryonic development, adult tissue homeostasis, and regeneration. Alterations in the cellular response to TGFβ are involved in severe human diseases. To understand how cells encode the extracellular input and transmit its information to elicit appropriate responses, we acquired quantitative time‐resolved measurements of pathway activation at the single‐cell level. We established dynamic time warping to quantitatively compare signaling dynamics of thousands of individual cells and described heterogeneous single‐cell responses by mathematical modeling. Our combined experimental and theoretical study revealed that the response to a given dose of TGFβ is determined cell specifically by the levels of defined signaling proteins. This heterogeneity in signaling protein expression leads to decomposition of cells into classes with qualitatively distinct signaling dynamics and phenotypic outcome. Negative feedback regulators promote heterogeneous signaling, as a SMAD7 knock‐out specifically affected the signal duration in a subpopulation of cells. Taken together, we propose a quantitative framework that allows predicting and testing sources of cellular signaling heterogeneity.     

Regulatory mechanisms and function of ERK MAP kinases

Spatiotemporal control of the Ras/ERK MAP kinase signaling pathway is a key factor for determining the specificity of cellular responses including cell proliferation, cell differentiation and cell survival. The fidelity of this signaling is regulated by docking interactions as well as scaffolding. Subcellular localization of ERK is controlled by cytoplasmic ERK anchoring proteins that have a nuclear export signal (NES), such as MEK. In quiescent cells, ERK and MEK localize to the cytoplasm. In response to stimulation, dissociation of the MEK-ERK complex is induced and activated ERK translocates to the nucleus. Recently, several negative regulators for Ras/ERK signaling have been identified and their detailed molecular mechanisms have been analyzed. Among them, Sprouty and Sef act as a temporal and a spatial regulator, respectively, for Ras/ERK signaling. Thus, multiple factors are involved in control of Ras/ERK signaling.     

Emergence of bimodal cell population responses from the interplay between analog single-cell signaling and protein expression noise

ABSTRACT: BACKGROUND: Cell-to-cell variability in protein expression can be large, and its propagation through signaling networks affects biological outcomes. Here, we apply deterministic and probabilistic models and biochemical measurements to study how network topologies and cell-to-cell protein abundance variations interact to shape signaling responses. RESULTS: We observe bimodal distributions of extracellular signal-regulated kinase (ERK) responses to epidermal growth factor (EGF) stimulation, which are generally thought to indicate bistable or ultrasensitive signaling behavior in single cells. Surprisingly, we find that a simple MAPK/ERK-cascade model with negative feedback that displays graded, analog ERK responses at a single cell level can explain the experimentally observed bimodality at the cell population level. Model analysis suggests that a conversion of graded input--output responses in single cells to digital responses at the population level is caused by a broad distribution of ERK pathway activation thresholds brought about by cell-to-cell variability in protein expression. CONCLUSIONS: Our results show that bimodal signaling response distributions do not necessarily imply digital (ultrasensitive or bistable) single cell signaling, and the interplay between protein expression noise and network topologies can bring about digital population responses from analog single cell dose responses. Thus, cells can retain the benefits of robustness arising from negative feedback, while simultaneously generating population-level on/off responses that are thought to be critical for regulating cell fate decisions.     

Epidermal growth factor and ionizing radiation up-regulate the DNA repair genes XRCC1 and ERCC1 in DU145 and LNCaP prostate carcinoma through MAPK signaling

This work examined the importance of radiation-induced and ligand-induced EGFR-ERK signaling for the regulation of DNA repair proteins XRCC1 and ERCC1 in prostate carcinoma cells, DU145 (TP53(mut)), displaying EGFR-TGFA-dependent autocrine growth and high MAPK (ERK1/2) activity, and LNCaP (TP53(wt)) cells expressing low constitutive levels of ERK1/2 activity. Using quantitative RT-PCR and Western analyses, we determined that ionizing radiation activated the DNA repair genes XRCC1 and ERCC1 in an ERK1/2-dependent fashion for each cell line. After irradiation, a rapid increase followed by a decrease in ERK1/2 activity preceded the increase in XRCC1/ERCC1 expression in DU145 cells, while only the rapid decrease in ERK1/2 preceded the increase in XRCC1/ERCC1 expression in LNCaP cells. Administration of EGF, however, markedly increased the up-regulation of phospho-ERK, ERCC1 and XRCC1 in both cell lines. Although the EGFR inhibitor tyrphostin (AG-1478) and the MEK inhibitor PD90859 both attenuated EGF-induced levels of the ERCC1 and XRCC1 protein, PD98059 blocked the induction of ERCC1 and XRCC1 by radiation more effectively in both cell lines. Inhibition of ERK at a level that reduced the up-regulation of DNA repair led to the persistence of apurinic/apyrimidinic (AP) sites of DNA damage and increased cell killing. Taken together, these data imply a complex control of DNA repair activation that may be more generally dependent on MAPK (ERK1/2) signaling than was previously noted. These data provide novel insights into the capacity of the EGFR-ERK signaling to modulate DNA repair in cancer cells and into the functional significance of this signaling.     

Suppression of MAPK signaling in BRAF‐activated PTEN‐deficient melanoma by blocking β‐catenin signaling in cancer‐associated fibroblasts

Cancer‐associated fibroblasts (CAFs) in the tumor microenvironment have been associated with formation of a dynamic and optimized niche for tumor cells to grow and evade cell death induced by therapeutic agents. We recently reported that ablation of β‐catenin expression in stromal fibroblasts and CAFs disrupted their biological activities in in vitro studies and in an in vivo B16F10 mouse melanoma model. Here, we show that the development of a BRAF‐activated PTEN‐deficient mouse melanoma was significantly suppressed in vivo after blocking β‐catenin signaling in CAFs. Further analysis revealed that expression of phospho‐Erk1/2 and phospho‐Akt was greatly reduced, effectively abrogating the activating effects and abnormal cell cycle progression induced by Braf and Pten mutations. In addition, the epithelial–mesenchymal transition (EMT)‐like process was also suppressed in melanoma cells. Taken together, our data highlight an important crosstalk between CAFs and the RAF‐MEK‐ERK signaling cascade in BRAF‐activated melanoma and may offer a new approach to abrogate host‐dependent drug resistance in targeted therapy.     

TCR‐β repertoire analysis of antigen‐specific single T cells using a high‐density microcavity array

The antigen specificity of cytotoxic T cells, provided by T‐cell receptors (TCRs), plays a central role in human autoimmune diseases, infection, and cancer. As the TCR repertoire is unique in individual cytotoxic T cells, a strategy to analyze its gene rearrangement at the single‐cell level is required. In this study, we applied a high‐density microcavity array enabling target cell screening of several thousands of single cells for identification of functional TCR‐β gene repertoires specific to melanoma (gp100) and cytomegalovirus (CMV) antigens. T cells expressing TCRs with the ability to recognize fluorescent‐labeled antigen peptide tetramers were isolated by using a micromanipulator under microscopy. Regularly arranged cells on the microcavity array eased detection and isolation of target single cells from a polyclonal T‐cell population. The isolated single cells were then directly utilized for RT‐PCR. By sequencing the amplified PCR products, antigen‐specific TCR‐β repertoires for gp100 and human cytomegalovirus antigens were successfully identified at the single‐cell level. This simple, accurate, and cost‐effective technique for single‐cell analysis has further potential as a valuable and widely applicable tool for studies on gene screening and expression analyses of various kinds of cells. Biotechnol. Bioeng. 2010;106: 311–318. © 2010 Wiley Periodicals, Inc.     

IGFBP7 regulates sepsis-induced acute kidney injury through ERK1/2 signaling

IGFBP7 as an early biomarker has been used to identify patients at risk of developing acute kidney injury (AKI). Nevertheless, its role in AKI remains obscure. The aim of our study is to determine the role and mechanism of IGFBP7 in lipopolysaccharide (LPS)-induced HK-2 cells in vitro and on sepsis-induced AKI by cecal ligation and puncture (CLP) in vivo. Here, we identified that IGFBP7 expression was increased in patients with AKI and HK-2 cells with LPS (1, 2, and 5 μg/mL) induction. HK-2 cells with LPS induction showed cell cycle arrest at G1-G0 phases and cell apoptosis and activated ERK1/2 parallel with the changes in the proteins belonging to the ERK1/2 pathway, including Cyclin D1, P21, Bax, and Bcl-2, which were inhibited by the IGFBP7 knockdown. Moreover, IGFBP7 overexpression significantly induced cell cycle arrest at G1-G0 phases and cell apoptosis of HK-2 cells, which were inhibited by PD98509, an ERK1/2 signaling inhibitor. IGFBP7 knockdown effectively alleviated the severity of the renal injury, evidenced by decreases in the urinary levels of creatinine, blood urea nitrogen, and albumin, cell apoptosis, and activation of ERK1/2 signaling in CLP mice. Taken together, our findings indicate that IGFBP7 regulates sepsis-induced AKI through ERK1/2 signaling.     

Assessing seasonal demographic covariation to understand environmental‐change impacts on a hibernating mammal

Natural populations are exposed to seasonal variation in environmental factors that simultaneously affect several demographic rates (survival, development and reproduction). The resulting covariation in these rates determines population dynamics, but accounting for its numerous biotic and abiotic drivers is a significant challenge. Here, we use a factor‐analytic approach to capture partially unobserved drivers of seasonal population dynamics. We use 40 years of individual‐based demography from yellow‐bellied marmots (Marmota flaviventer) to fit and project population models that account for seasonal demographic covariation using a latent variable. We show that this latent variable, by producing positive covariation among winter demographic rates, depicts a measure of environmental quality. Simultaneously, negative responses of winter survival and reproductive‐status change to declining environmental quality result in a higher risk of population quasi‐extinction, regardless of summer demography where recruitment takes place. We demonstrate how complex environmental processes can be summarized to understand population persistence in seasonal environments.