Integrins and EGFR coordinately regulate the pro-apoptotic protein Bim to prevent anoikis

Epithelial cells must adhere to the extracellular matrix (ECM) for survival, as detachment from matrix triggers apoptosis or anoikis. Integrins are major mediators of adhesion between cells and ECM proteins, and transduce signals required for cell survival. Recent evidence suggests that integrin receptors are coupled to growth factor receptors in the regulation of multiple biological functions; however, mechanisms involved in coordinate regulation of cell survival are poorly understood and mediators responsible for anoikis have not been well characterized. Here, we identify the pro-apoptotic protein Bim as a critical mediator of anoikis in epithelial cells. Bim is strongly induced after cell detachment and downregulation of Bim expression by RNA interference (RNAi) inhibits anoikis. Detachment-induced expression of Bim requires a lack of beta(1)-integrin engagement, downregulation of EGF receptor (EGFR) expression and inhibition of Erk signalling. Overexpressed EGFR was uncoupled from integrin regulation, resulting in the maintenance of Erk activation in suspension, and a block in Bim expression and anoikis. Thus, Bim functions as a key sensor of integrin and growth factor signals to the Erk pathway, and loss of such coordinate regulation may contribute to tumour progression.     

Mesothelin promotes anchorage-independent growth and prevents anoikis via extracellular signal-regulated kinase signaling pathway in human breast cancer cells

Mesothelin (MSLN) is a glycoprotein that is overexpressed in various tumors. MSLN is present on the cell surface and is also released into body fluids or culture supernatants from MSLN-positive tumor cells. Despite intensive study of MSLN as a diagnostic marker or target for immunotherapy, its biological function is largely unknown. In the present study, we examined the effects of ectopic expression of MSLN in human breast cancer cell lines (MCF-7, T47D, and MDA-MB-231). We found that overexpression of MSLN promoted anchorage-independent growth in soft agar. In addition, MDA-MB-231 cells expressing high levels of MSLN exhibited resistance to anoikis (a type of apoptosis induced by detachment from substratum), as indicated by decreased DNA fragmentation and down-regulation of the proapoptotic protein Bim. Incubating MSLN-expressing MDA-MB-231 cells in the presence of U0126, an inhibitor of mitogen-activated protein/extracellular-signal-regulated kinase kinase, induced accumulation of Bim and restored susceptibility to anoikis. Western blot analysis also revealed that overexpression of MSLN resulted in sustained activation of extracellular signal-regulated kinase 1/2 and suppression of Bim. The present results constitute novel evidence that MSLN enables cells to survive under anchorage-independent conditions by suppressing Bim induction via the extracellular signal-regulated kinase signaling pathway.     

Matrix attachment regulates Fas-induced apoptosis in endothelial cells: a role for c-flip and implications for anoikis

Survival of endothelial cells is critical for cellular processes such as angiogenesis. Cell attachment to extracellular matrix inhibits apoptosis in endothelial cells both in vitro and in vivo, but the molecular mechanisms underlying matrix-induced survival signals or detachment-induced apoptotic signals are unknown. We demonstrate here that matrix attachment is an efficient regulator of Fas-mediated apoptosis in endothelial cells. Thus, matrix attachment protects cells from Fas-induced apoptosis, whereas matrix detachment results in susceptibility to Fas-mediated cell death. Matrix attachment modulates Fas-mediated apoptosis at two different levels: by regulating the expression level of Fas, and by regulating the expression level of c-Flip, an endogenous antagonist of caspase-8. The extracellular signal-regulated kinase (Erk) cascade functions as a survival pathway in adherent cells by regulating c-Flip expression. We further show that detachment-induced cell death, or anoikis, itself results from activation of the Fas pathway by its ligand, Fas-L. Fas-L/Fas interaction, Fas-FADD complex formation, and caspase-8 activation precede the bulk of anoikis in endothelial cells, and inhibition of any of these events blocks anoikis. These studies identify matrix attachment as a survival factor against death receptor-mediated apoptosis and provide a molecular mechanism for anoikis and previously observed Fas resistance in endothelial cells.     

A discrete cell cycle checkpoint in late G(1) that is cytoskeleton-dependent and MAP kinase (Erk)-independent

Cell spreading on extracellular matrix and associated changes in the actin cytoskeleton (CSK) are necessary for progression through G(1) and entry into S phase of the cell cycle. Pharmacological disruption of CSK integrity inhibits early mitogenic signaling to the extracellular signal-regulated kinase (Erk) subfamily of the mitogen-activated protein kinases (MAPKs) and arrests the cell cycle in G(1). Here we show that this block of G(1) progression is not simply a consequence of inhibition of the MAPK/Erk pathway but instead it reveals the existence of a discrete CSK-sensitive checkpoint. Use of PD98059 to inhibit MAPK/Erk and cytochalasin D (Cyto D) to disrupt the actin CSK at progressive time points in G(1) revealed that the requirement for MAPK/Erk activation lasts only to mid-G(1), while the actin CSK must remain intact up to late G(1) restriction point, R, in order for capillary endothelial cells to enter S phase. Additional analysis using Cyto D pulses defined a narrow time window of 3 h just prior to R in which CSK integrity was shown to be critical for the G(1)/S transition. Cyto D treatment led to down-regulation of cyclin D1 protein and accumulation of the cdk inhibitor, p27(Kip1), independent of cell cycle phase, suggesting that these changes resulted directly from CSK disruption rather than from a general cell cycle block. Together, these data indicate the existence of a distinct time window in late G(1) in which signals elicited by the CSK act independently of early MAPK/Erk signals to drive the cell cycle machinery through the G(1)/S boundary and, hence, promote cell growth.     

Erk5 is activated and acts as a survival factor in mitosis

Erk5 is a recently discovered MAPK claimed to be responsible for some of the roles attributed to Erk1/2; here we report that it is activated in mitosis in comparison to G1/S. When Erk5 is inactivated pharmacologically or largely ablated by RNAi, cell survival in mitosis is diminished. We have previously shown Bim, a BH3-only protein of the Bcl-2 family, to be phosphorylated in mitosis, in a MEK-dependent manner (M. Grãos, A. D. Almeida, S. Chatterjee, Biochem. J. 388 (2005) 185). Inactivation of Erk5 in mitosis causes dephosphorylation of Bim. Bim is in the mitochondria in mitosis and when dephosphorylated interacts with Bax, inducing caspase activation. We also show that in mitosis Bim co-immunoprecipitates with Erk5 and Erk5 phosphorylates GST-Bim in in vitro kinase reaction. Taken together, our results identify a new target of the still largely mysterious Erk5 and suggest that Erk5 in mitosis may be a decisive step for the survival of proliferating cells.     

Human intestinal epithelial crypt cell survival and death: Complex modulations of Bcl-2 homologs by Fak, PI3-K/Akt-1, MEK/Erk, and p38 signaling pathways

To investigate the mechanisms responsible for survival and apoptosis/anoikis in normal human intestinal epithelial crypt cells, we analyzed the roles of various signaling pathways and cell adhesion on the expression of six Bcl-2 homologs (Bcl-2, Bcl-XL, Mcl-1, Bax, Bak, Bad) in the well established HIEC-6 cell model. Pharmacological inhibitors and/or dominant-negative constructs were used to inhibit focal adhesion kinase (Fak) and p38 isoforms, as well as the phosphatidylinositol 3'-kinase (PI3-K)/Akt-1 and mitogen-activated protein kinase [MAPK] kinase (MEK)/extracellular regulated kinases (Erk) pathways. Cell adhesion was disrupted by antibody-inhibition of integrin binding or forced cell suspension. The activation levels of studied kinase pathways were also analyzed. Herein, we report that beta1 integrins, Fak, and the PI3-K/Akt-1 pathway, but not beta4 integrins or the MEK/Erk pathway, are crucial for the survival of HIEC-6 cells. Conversely, p38beta, but not p38alpha or gamma, is required for the induction of apoptosis/anoikis in HIEC-6 cells. However, each of the signaling molecules/pathways analyzed were found to affect distinctively the individual expression of the Bcl-2 homologs studied. For example, the inhibition of the PI3-K/Akt-1 pathway down-regulated Bcl-XL, Mcl-1, and Bad, while at the same time up-regulating Bax, whereas the inhibition of Fak up-regulated both Bax and Bak, down-regulated Bad, and did not affect the other Bcl-2 homologs analyzed. These results indicate that integrins, Fak, PI3-K/Akt-1, MEK/Erk, and p38 isoforms perform distinct roles in the regulation of HIEC-6 cell survival and/or death. In addition, our data show that the functions performed by these molecules/pathways in promoting cell survival or apoptosis/anoikis translate into complex, differential modulations of individual Bcl-2 homologs.     

Roles of the Raf/MEK/ERK pathway in cell growth, malignant transformation and drug resistance

Growth factors and mitogens use the Ras/Raf/MEK/ERK signaling cascade to transmit signals from their receptors to regulate gene expression and prevent apoptosis. Some components of these pathways are mutated or aberrantly expressed in human cancer (e.g., Ras, B-Raf). Mutations also occur at genes encoding upstream receptors (e.g., EGFR and Flt-3) and chimeric chromosomal translocations (e.g., BCR-ABL) which transmit their signals through these cascades. Even in the absence of obvious genetic mutations, this pathway has been reported to be activated in over 50% of acute myelogenous leukemia and acute lymphocytic leukemia and is also frequently activated in other cancer types (e.g., breast and prostate cancers). Importantly, this increased expression is associated with a poor prognosis. The Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt pathways interact with each other to regulate growth and in some cases tumorigenesis. For example, in some cells, PTEN mutation may contribute to suppression of the Raf/MEK/ERK cascade due to the ability of activated Akt to phosphorylate and inactivate different Rafs. Although both of these pathways are commonly thought to have anti-apoptotic and drug resistance effects on cells, they display different cell lineage specific effects. For example, Raf/MEK/ERK is usually associated with proliferation and drug resistance of hematopoietic cells, while activation of the Raf/MEK/ERK cascade is suppressed in some prostate cancer cell lines which have mutations at PTEN and express high levels of activated Akt. Furthermore the Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt pathways also interact with the p53 pathway. Some of these interactions can result in controlling the activity and subcellular localization of Bim, Bak, Bax, Puma and Noxa. Raf/MEK/ERK may promote cell cycle arrest in prostate cells and this may be regulated by p53 as restoration of wild-type p53 in p53 deficient prostate cancer cells results in their enhanced sensitivity to chemotherapeutic drugs and increased expression of Raf/MEK/ERK pathway. Thus in advanced prostate cancer, it may be advantageous to induce Raf/MEK/ERK expression to promote cell cycle arrest, while in hematopoietic cancers it may be beneficial to inhibit Raf/MEK/ERK induced proliferation and drug resistance. Thus the Raf/MEK/ERK pathway has different effects on growth, prevention of apoptosis, cell cycle arrest and induction of drug resistance in cells of various lineages which may be due to the presence of functional p53 and PTEN and the expression of lineage specific factors.     

Fak/Src signaling in human intestinal epithelial cell survival and anoikis: differentiation state-specific uncoupling with the PI3-K/Akt-1 and MEK/Erk pathways

Human intestinal epithelial cell survival and anoikis are distinctively regulated according to the state of differentiation. In the present study, we analyzed the roles of focal adhesion kinase (Fak)/Src signaling to the PI3-K/Akt-1 and mitogen-activated protein kinase (MEK)/extracellular regulated kinases (Erk) pathways, within the context of such differentiation-state distinctions. Anoikis was induced by inhibition of beta1 integrins (antibody blocking), inhibition of Fak (pharmacologic inhibition or overexpression of dominant negative mutants), or by maintaining cells in suspension. Activation parameters of Fak, Src, Akt-1, and Erk1/2 were analyzed. Activities of Src, Akt-1, or Erk1/2 were also blocked by pharmacological inhibition or by overexpression of dominant-negative mutants. We report that: (1) the loss or inhibition of beta1 integrin binding activity causes anoikis and results in a down-activation of Fak, Src, Akt-1, and Erk1/2 in both undifferentiated, and differentiated cells; (2) the inhibition of Fak likewise causes anoikis and a down-activation of Src, Akt-1, and Erk1/2, regardless of the differentiation state; (3) Src, PI3-K/Akt-1, and MEK/Erk contribute to the survival of differentiated cells, whereas MEK/Erk does not play a role in the survival of undifferentiated ones; (4) the inhibition/loss of beta1 integrin binding and/or Fak activity results in a loss of Src engagement with Fak, regardless of the state of differentiation; and (5) Src contributes to the activation of both the PI3-K/Akt-1 and MEK/Erk pathways in undifferentiated cells, but does not influence PI3-K/Akt-1 in differentiated ones. Hence, Fak/Src signaling to the PI3-K/Akt-1 and MEK/Erk pathways undergoes a differentiation state-specific uncoupling which ultimately reflects upon the selective engagement of these same pathways in the mediation of intestinal epithelial cell survival.     

Extracellular matrix metalloproteinase inducer (CD147) confers resistance of breast cancer cells to Anoikis through inhibition of Bim

Overexpression of extracellular matrix metalloproteinase inducer (EMMPRIN or CD147), a member of the immunoglobulin family and a glycoprotein enriched on the surface of tumor cells, promotes invasion, metastasis, and growth and survival of malignant cells and confers resistance to some chemotherapeutic drugs. However, the molecular mechanisms underlying the actions of EMMPRIN are not fully understood. In this study we sought to determine whether EMMPRIN contributes to the malignant phenotype of breast cancer by inhibiting anoikis, a form of apoptosis induced by loss or alteration of cell-cell or cell-matrix anchorage, and to explore the signaling pathways involved. We found that in the absence of attachment, human breast carcinoma cells expressing high levels of EMMPRIN formed less compact aggregates with larger surface area and less fibronectin matrix assembly, had higher viability, and were resistant to anoikis. Knockdown of EMMPRIN expression by RNA interference (small interfering RNA or short hairpin RNA) sensitized cancer cells to anoikis, as demonstrated by activation of caspase-3, increased DNA fragmentation, and decreased cellular viability. Furthermore, we observed that the accumulation of Bim, a proapoptotic BH3-only protein, was reduced in EMMPRIN-expressing cells and that silencing of EMMPRIN expression elevated Bim protein levels and enhanced cellular sensitivity to anoikis. Treatment of cells with a MEK inhibitor (U0126) or proteasome inhibitor (epoxomicin) also up-regulated Bim accumulation and rendered cells more sensitive to anoikis. These results indicated that expression of EMMPRIN protects cancer cells from anoikis and that this effect is mediated at least in part by a MAP kinase-dependent reduction of Bim. Because anoikis deficiency is a key feature of neoplastic transformation and invasive growth of epithelial cancer cells, our study on the role of EMMPRIN in anoikis resistance and the mechanism involved underscores the potential of EMMPRIN expression as a prognostic marker and novel target for cancer therapy.     

Intestinal epithelial cancer cell anoikis resistance: EGFR‐mediated sustained activation of Src overrides Fak‐dependent signaling to MEK/Erk and/or PI3‐K/Akt‐1

Herein, we investigated the survival roles of Fak, Src, MEK/Erk, and PI3‐K/Akt‐1 in intestinal epithelial cancer cells (HCT116, HT29, and T84), in comparison to undifferentiated and differentiated intestinal epithelial cells (IECs). We report that: (1) cancer cells display striking anoikis resistance, as opposed to undifferentiated/differentiated IECs; (2) under anoikis conditions and consequent Fak down‐activation, cancer cells nevertheless exhibit sustained Fak–Src interactions and Src/MEK/Erk activation, unlike undifferentiated/differentiated IECs; however, HCT116 and HT29 cells exhibit a PI3‐K/Akt‐1 down‐activation, as undifferentiated/differentiated IECs, whereas T84 cells do not; (3) cancer cells require MEK/Erk for survival, as differentiated (but not undifferentiated) IECs; however, T84 cells do not require Fak and HCT116 cells do not require PI3‐K/Akt‐1, in contrast to the other cells studied; (4) Src acts as a cornerstone in Fak‐mediated signaling to MEK/Erk and PI3‐K/Akt‐1 in T84 cells, as in undifferentiated IECs, whereas PI3‐K/Akt‐1 is Src‐independent in HCT116, HT29 cells, as in differentiated IECs; and (5) EGFR activity inhibition abrogates anoikis resistance in cancer cells through a loss of Fak–Src interactions and down‐activation of Src/MEK/Erk (T84, HCT116, HT29 cells) and PI3‐K/Akt‐1 (T84 cells). Hence, despite distinctions in signaling behavior not necessarily related to undifferentiated or differentiated IECs, intestinal epithelial cancer cells commonly display an EGFR‐mediated sustained activation of Src under anoikis conditions. Furthermore, such sustained Src activation confers anoikis resistance at least in part through a consequent sustenance of Fak–Src interactions and MEK/Erk activation, thus not only overriding Fak‐mediated signaling to MEK/Erk and/or PI3‐K/Akt‐1, but also the requirement of Fak and/or PI3‐K/Akt‐1 for survival. J. Cell. Biochem. 107: 639–654, 2009. © 2009 Wiley‐Liss, Inc.     

The p42/p44 mitogen-activated protein kinase activation triggers p27Kip1 degradation independently of CDK2/cyclin E in NIH 3T3 cells.

The p42/p44 mitogen-activated protein (MAP) kinase is stimulated by various mitogenic stimuli, and its sustained activation is necessary for cell cycle G(1) progression and G(1)/S transition. G(1) progression and G(1)/S transition also depend on sequential cyclin-dependent kinase (CDK) activation. Here, we demonstrate that MAP kinase inhibition leads to accumulation of the CDK inhibitor p27(Kip1) in NIH 3T3 cells. Blocking the proteasome-dependent degradation of p27(Kip1) impaired this accumulation, suggesting that MAP kinase does not act on p27(Kip1) protein synthesis. In the absence of extracellular signals (growth factors or cell adhesion), genetic activation of MAP kinase decreased the expression of p27(Kip1) as assessed by cotransfection experiments and by immunofluorescence detection. Importantly, MAP kinase activation also decreased the expression of a p27(Kip1) mutant, which cannot be phosphorylated by CDK2, suggesting that MAP kinase-dependent p27(Kip1) regulation is CDK2-independent. Accordingly, expression of dominant-negative CDK2 did not impair the down-regulation of p27(Kip1) induced by MAP kinase activation. These data demonstrate that the MAP kinase pathway regulates p27(Kip1) expression in fibroblasts essentially through a degradation mechanism, independently of p27(Kip1) phosphorylation by CDK2. This strengthens the role of this CDK inhibitor as a key effector of G(1) growth arrest, whose expression can be controlled by extracellular stimuli-dependent signaling pathways.     

SOCS1 counteracts ROS-mediated survival signals and promotes apoptosis by modulating cell cycle to increase radiosensitivity of colorectal cancer cells

As negative regulators of cytokine signaling pathways, suppressors of cytokine signaling (SOCS) proteins have been reported to possess both pro-tumor and anti-tumor functions. Our recent studies have demonstrated suppressive effects of SOCS1 on epithelial to mesenchymal signaling in colorectal cancer cells in response to fractionated ionizing radiation or oxidative stress. The objective of the present study was to determine the radiosensitizing action of SOCS1 as an anti-tumor mechanism in color-ectal cancer cell model. In HCT116 cells exposed to ionizing radiation, SOCS1 over-expression shifted cell cycle arrest from G2/M to G1 and promoted radiation-induced apoptosis in a p53-dependent manner with down-regulation of cyclin B and up-regulation of p21. On the other hand, SOCS1 knock-down resulted in a reduced apoptosis with a decrease in G1 arrest. The regulatory action of SOCS1 on the radiation response was mediated by inhibition of radiation-induced Jak3/STAT3 and Erk activities, thereby blocking G1 to S transition. Radiation-induced early ROS signal was responsible for the activation of Jak3/Erk/STAT3 that led to cell survival response. Our data col-lectively indicate that SOCS1 can promote radiosensitivity of colorectal cancer cells by counteracting ROS-mediated survival signal, thereby blocking cell cycle progression from G1 to S. The resulting increase in G1 arrest with p53 activation then contributes to the promotion of apoptotic response upon radiation. Thus, induction of SOCS1 expression may increase therapeutic efficacy of radiation in tumors with low SOCS1 levels.     

Oncogenic Ras Blocks Anoikis by Activation of a Novel Effector Pathway Independent of Phosphatidylinositol 3-Kinase

Activated Ras, but not Raf, causes transformation of RIE-1 rat intestinal epithelial cells, demonstrating the importance of Raf-independent effector signaling in mediating Ras transformation. To further assess the contribution of Raf-dependent and Raf-independent function in oncogenic Ras transformation, we evaluated the mechanism by which oncogenic Ras blocks suspension-induced apoptosis, or anoikis, of RIE-1 cells. We determined that oncogenic versions of H-, K-, and N-Ras, as well as the Ras-related proteins TC21 and R-Ras, protected RIE-1 cells from anoikis. Surprisingly, our analyses of Ras effector domain mutants or constitutively activated effectors indicated that activation of Raf-1, phosphatidylinositol 3-kinase (PI3K), or RalGDS alone is not sufficient to promote Ras inhibition of anoikis. Treatment of Ras-transformed cells with the U0126 MEK inhibitor caused partial reversion to an anoikis-sensitive state, indicating that extracellular signal-regulated kinase activation contributes to inhibition of anoikis. Unexpectedly, oncogenic Ras failed to activate Akt, and treatment of Ras-transformed RIE-1 cells with the LY294002 PI3K inhibitor did not affect anoikis resistance or growth in soft agar. Thus, while important for Ras transformation of fibroblasts, PI3K may not be involved in Ras transformation of RIE-1 cells. Finally, inhibition of epidermal growth factor receptor kinase activity did not overcome Ras inhibition of anoikis, indicating that this autocrine loop essential for transformation is not involved in anoikis protection. We conclude that a PI3K- and RalGEF-independent Ras effector(s) likely cooperates with Raf to confer anoikis resistance upon RIE-1 cells, thus underscoring the complex nature by which Ras transforms cells.     

Intrinsically active variants of Erk oncogenically transform cells and disclose unexpected autophosphorylation capability that is independent of TEY phosphorylation

The receptor-tyrosine kinase (RTK)/Ras/Raf pathway is an essential cascade for mediating growth factor signaling. It is abnormally overactive in almost all human cancers. The downstream targets of the pathway are members of the extracellular regulated kinases (Erk1/2) family, suggesting that this family is a mediator of the oncogenic capability of the cascade. Although all oncogenic mutations in the pathway result in strong activation of Erks, activating mutations in Erks themselves were not reported in cancers. Here we used spontaneously active Erk variants to check whether Erk’s activity per se is sufficient for oncogenic transformation. We show that Erk1(R84S) is an oncoprotein, as NIH3T3 cells that express it form foci in tissue culture plates, colonies in soft agar, and tumors in nude mice. We further show that Erk1(R84S) and Erk2(R65S) are intrinsically active due to an unusual autophosphorylation activity they acquire. They autophosphorylate the activatory TEY motif and also other residues, including the critical residue Thr-207 (in Erk1)/Thr-188 (in Erk2). Strikingly, Erk2(R65S) efficiently autophosphorylates its Thr-188 even when dually mutated in the TEY motif. Thus this study shows that Erk1 can be considered a proto-oncogene and that Erk molecules possess unusual autoregulatory properties, some of them independent of TEY phosphorylation.     

The novel agent ophiobolin O induces apoptosis and cell cycle arrest of MCF-7 cells through activation of MAPK signaling pathways

Ophiobolin O is a natural compound that has been isolated from Aspergillus ustus 094102. This is the first study to demonstrate the anti-proliferative effect of ophiobolin O in human breast cancer MCF-7 cells. The results of present study show that ophiobolin O induced cycle G(0)/G(1) phase arrest in MCF-7 cells using a cell cycle analysis. In addition, we demonstrated that ophiobolin O reduced the viability of human breast cancer MCF-7 cells in a time- and dose-dependent manner and efficiently induced apoptosis in MCF-7 cells using the Annexin V/PI binding assay. Ophiobolin O also caused the activation of JNK (c-Jun NH(2)-terminal kinase), p38 MAPK (mitogen activated protein kinase) and ERK (extracellular signal-regulated kinase) as well as the degradation of Bcl-2 phosphorylation (Ser70). Bax protein expression was not changed in ophiobolin O-treated cells. Taken together, ophiobolin O may be considered as a novel therapeutic agent in breast cancer.     

Redox regulation of anoikis: reactive oxygen species as essential mediators of cell survival

Proper attachment to the extracellular matrix (ECM) is essential for cell survival. The loss of integrin-mediated cell-ECM contact results in an apoptotic process termed anoikis. However, mechanisms involved in regulation of cell survival are poorly understood and mediators responsible for anoikis have not been well characterized. Here, we demonstrate that reactive oxygen species (ROS) produced through the involvement of the small GTPase Rac-1 upon integrin engagement exert a mandatory role in transducing a pro-survival signal that ensures that cells escape from anoikis. In particular, we show that ROS are responsible for the redox-mediated activation of Src that trans-phosphorylates epidermal growth factor receptor (EGFR) in a ligand-independent manner. The redox-dependent phosphorylation of EGFR activates both extracellular signal-regulated protein kinase and Akt downstream signalling pathways, culminating in degradation of the pro-apoptotic protein Bim. Hence, our results shed new light on the mechanism granting the adhesion-dependent antiapoptotic effect, highlighting a fundamental role of ROS-mediated Src regulation in ensuring anoikis protection.     

Lack of Correlation between Activation of Jun–NH2-terminal Kinase and Induction of Apoptosis after Detachment of Epithelial Cells

Detachment of epithelial cells from the extracellular matrix leads to induction of programmed cell death, a process that has been termed “anoikis.” It has been reported recently that detachment of MDCK cells from matrix results in activation of Jun–NH₂-terminal kinases (JNKs) and speculated that these stress activated protein kinases play a causal role in the induction of anoikis (Frisch, S.M., K. Vuori, D. Kelaita, and S. Sicks. 1996. J. Cell Biol. 135:1377–1382). We report here that although JNK is activated by detachment of normal MDCK cells, study of cell lines expressing activated signaling proteins usually controlled by Ras shows that stimulation of JNK fails to correlate with induction of anoikis. Activated phosphoinositide 3-OH kinase and activated PKB/Akt protect MDCK cells from detachment-induced apoptosis without suppressing JNK activation. Conversely, activated Raf and dominant negative SEK1, a JNK kinase, attenuate detachment-induced JNK activation without protecting from apoptosis. zVAD-fmk, a peptide inhibitor of caspases, prevents MDCK cell anoikis without affecting JNK activation. p38, a related stress-activated kinase, is also stimulated by detachment from matrix, but inhibition of this kinase with SB 203580 does not protect from anoikis. It is therefore unlikely that either JNK or p38 play a direct role in detachment-induced programmed cell death in epithelial cells.     

Raf-induced proliferation or cell cycle arrest is determined by the level of Raf activity with arrest mediated by p21Cip1.

The Raf family of protein kinases display differences in their abilities to promote the entry of quiescent NIH 3T3 cells into the S phase of the cell cycle. Although conditional activation of deltaA-Raf:ER promoted cell cycle progression, activation of deltaRaf-1:ER and deltaB-Raf:ER elicited a G1 arrest that was not overcome by exogenously added growth factors. Activation of all three deltaRaf:ER kinases led to elevated expression of cyclin D1 and cyclin E and reduced expression of p27Kip1. However, activation of deltaB-Raf:ER and deltaRaf-1:ER induced the expression of p21Cip1, whereas activation of deltaA-Raf:ER did not. A catalytically potentiated form of deltaA-Raf:ER, generated by point mutation, strongly induced p21Cip1 expression and elicited cell cycle arrest similarly to deltaB-Raf:ER and deltaRaf-1:ER. These data suggested that the strength and duration of signaling by Raf kinases might influence the biological outcome of activation of this pathway. By titration of deltaB-Raf:ER activity we demonstrated that low levels of Raf activity led to activation of cyclin D1-cdk4 and cyclin E-cdk2 complexes and to cell cycle progression whereas higher Raf activity elicited cell cycle arrest correlating with p21Cip1 induction and inhibition of cyclin-cdk activity. Using green fluorescent protein-tagged forms of deltaRaf-1:ER in primary mouse embryo fibroblasts (MEFs) we demonstrated that p21Cip1 was induced by Raf in a p53-independent manner, leading to cell cycle arrest. By contrast, activation of Raf in p21Cip1(-/-) MEFs led to a robust mitogenic response that was similar to that observed in response to platelet-derived growth factor. These data indicate that, depending on the level of kinase activity, Raf can elicit either cell cycle progression or cell cycle arrest in mouse fibroblasts. The ability of Raf to elicit cell cycle arrest is strongly associated with its ability to induce the expression of the cyclin-dependent kinase inhibitor p21Cip1 in a manner that bears analogy to alpha-factor arrest in Saccharomyces cerevisiae. These data are consistent with a role for Raf kinases in both proliferation and differentiation of mammalian cells.