Transforming growth factor-alpha prevents detachment-induced inhibition of c-Src kinase activity, Bcl-XL down-regulation, and apoptosis of intestinal epithelial cells

Detachment of epithelial cells from the extracellular matrix (ECM) results in apoptosis, a phenomenon often referred to as anoikis. Acquisition of anoikis resistance is now thought to be a prerequisite for the progression of carcinomas. Colorectal cancer cells frequently secrete epidermal growth factor receptor (EGFR) ligands, which are known to have anti-apoptotic activity. However, whether these ligands have the ability to inhibit anoikis of intestinal epithelial cells is unclear, since at least in some cell types efficient EGFR signaling requires cell-ECM adhesion. Here we report that transforming growth factor-alpha (TGF-alpha), an EGFR ligand that is frequently secreted by colorectal cancer cells, strongly inhibits anoikis of the non-malignant rat intestinal epithelial cell lines, IEC-18 and RIE-1. TGF-alpha exerts its anti-anoikis effect by preventing detachment-induced inhibition of c-Src kinase activity. We also show that Fas activation, a molecular event known to play a critical role in anoikis, is not suppressed by TGF-alpha. On the other hand, this growth factor strongly inhibits the detachment-induced down-regulation of Bcl-X(L), another change that is involved in the induction of anoikis. We further demonstrate that this inhibition occurs in a c-Src-dependent manner. We conclude that TGF-alpha has the ability to suppress anoikis of intestinal epithelial cells, at least in part, by reverting the loss of c-Src activity and Bcl-X(L) expression induced by detachment from the ECM.     

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.     

Functional role of death-associated protein 3 (DAP3) in anoikis

Detachment of adherent epithelial cells from the extracellular matrix induces apoptosis, known as anoikis. Integrin stimulation protects cells from anoikis, but the responsible mechanisms are not well known. Here, we demonstrated that a pro-apoptotic GTP-binding protein, DAP3 (death-associated protein 3), is critical for induction of anoikis. Down-regulation of DAP3 expression by antisense oligonucleotides inhibited anoikis. Conversely, overexpression of DAP3 augmented cell death and caspase activation induced by cell detachment. Furthermore, the association of DAP3 with FADD and the activation of caspase-8 were induced by cell detachment. We also showed that DAP3 is phosphorylated by kinase Akt (PKB), and active Akt can nullify apoptosis induction by DAP3. Mutation of a consensus Akt phosphorylation site in DAP3 renders it resistant to suppression by active Akt in cells. Integrin ligation stimulates Akt activation and phosphorylation of DAP3 in intact cells, as well as suppresses the ability of DAP3 overexpression to augment anoikis. Involvement of DAP3 in anoikis signaling demonstrates a novel role for this GTP-binding protein in apoptosis induction caused by cell detachment.     

Extracellular matrix regulation of autophagy

Integrin-mediated attachment of epithelial cells to extracellular matrix (ECM) is crucial for proper growth and survival. Although detachment leads to apoptosis, termed anoikis, recent work demonstrates that ECM detachment also robustly induces autophagy, a tightly regulated lysosomal self-digestion process that actually promotes survival. Autophagy presumably protects epithelial cells from the stresses of ECM detachment, allowing them to survive provided that they reattach in a timely manner. Currently, the intracellular signals linking integrin engagement to autophagy remain unclear, but certain growth factor, energy-sensing, and stress-response pathways represent attractive candidates. Moreover, autophagy may be a previously unrecognized mechanism utilized by detached cancer cells to survive anoikis, which may facilitate tumor cell dormancy, dissemination, and metastasis.     

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.     

Involvement of the ERK signaling cascade in protein kinase C-mediated cell cycle arrest in intestinal epithelial cells

We have reported previously that protein kinase C (PKC) signaling can mediate a program of cell cycle withdrawal in IEC-18 nontransformed intestinal crypt cells, involving rapid disappearance of cyclin D1, increased expression of Cip/Kip cyclin-dependent kinase inhibitors, and activation of the growth suppressor function of pocket proteins. In the current study, we present evidence to support a requisite role for PKC alpha in mediating these effects. Furthermore, analysis of the signaling events linking PKC/PKC alpha activation to changes in the cell cycle regulatory machinery implicate the Ras/Raf/MEK/ERK cascade. PKC/PKC alpha activity promoted GTP loading of Ras, activation of Raf-1, and phosphorylation/activation of ERK. ERK activation was found to be required for critical downstream effects of PKC/PKC alpha activation, including cyclin D1 down-regulation, p21(Waf1/Cip1) induction, and cell cycle arrest. PKC-induced ERK activation was strong and sustained relative to that produced by proliferative signals, and the growth inhibitory effects of PKC agonists were dominant over proliferative events when these opposing stimuli were administered simultaneously. PKC signaling promoted cytoplasmic and nuclear accumulation of ERK activity, whereas growth factor-induced phospho-ERK was localized only in the cytoplasm. Comparison of the effects of PKC agonists that differ in their ability to sustain PKC alpha activation and growth arrest in IEC-18 cells, together with the use of selective kinase inhibitors, indicated that the length of PKC-mediated cell cycle exit is dictated by the magnitude/duration of input signal (i.e. PKC alpha activity) and of activation of the ERK cascade. The extent/duration of phospho-ERK nuclear localization may also be important determinants of the duration of PKC agonist-induced growth arrest in this system. Taken together, the data point to PKC alpha and the Ras/Raf/MEK/ERK cascade as key regulators of cell cycle withdrawal in intestinal epithelial cells.     

Epstein-Barr Virus Latent Membrane Protein 2A Contributes to Anoikis Resistance through ERK Activation

Epstein-Barr virus (EBV) is associated with various malignancies, including epithelial cancers. In this study, we analyzed the effect of EBV infection on epithelial cells by using EBV-converted epithelial cells. In EBV-positive cells, the extracellular signal-regulated kinase (ERK) pathway is constitutively activated. Inhibition of ERK activity leads to reduced anoikis resistance; therefore, EBV-positive cells are more resistant to anoikis, a type of apoptosis induced by cell detachment, than are EBV-negative cells. Among the viral genes expressed in EBV-positive cells, the latent membrane protein 2A (LMP2A) is responsible for induction of ERK-mediated anoikis resistance, although the expression level of LMP2A is much lower in EBV-positive cells than in EBV-transformed B cells. Further analysis demonstrated that LMP2A downregulation of the proanoikis mediator Bim through proteasomal degradation is dependent on the immunoreceptor tyrosine-based activation motif (ITAM). These findings suggest that LMP2A-mediated ERK activation is involved in the generation of EBV-associated epithelial malignancies.     

IκB kinase complex (IKK) triggers detachment-induced autophagy in mammary epithelial cells independently of the PI3K-AKT-MTORC1 pathway

Adherent cells require proper integrin-mediated extracellular matrix (ECM) engagement for growth and survival; normal cells deprived of proper ECM contact undergo anoikis. At the same time, autophagy is induced as a survival pathway in both fibroblasts and epithelial cells upon ECM detachment. Here, we further define the intracellular signals that mediate detachment-induced autophagy and uncover an important role for the IκB kinase (IKK) complex in the induction of autophagy in mammary epithelial cells (MECs) deprived of ECM contact. Whereas the PI3K-AKT-MTORC1 pathway activation potently inhibits autophagy in ECM-detached fibroblasts, enforced activation of this pathway is not sufficient to suppress detachment-induced autophagy in MECs. Instead, inhibition of IKK, as well as its upstream regulator, MAP3K7/TAK1, significantly attenuates detachment-induced autophagy in MECs. Furthermore, function-blocking experiments corroborate that both IKK activation and autophagy induction result from decreased ITGA3-ITGB1 (α3β1 integrin) function. Finally, we demonstrate that pharmacological IKK inhibition enhances anoikis and accelerates luminal apoptosis during acinar morphogenesis in three-dimensional culture. Based on these results, we propose that the IKK complex functions as a key mediator of detachment-induced autophagy and anoikis resistance in epithelial cells.     

NF-kappa B-inducing kinase is a common mediator of IL-17-, TNF-alpha-, and IL-1 beta-induced chemokine promoter activation in intestinal epithelial cells

IL-17 expression is restricted to activated T cells, whereas the IL-17R is expressed in a variety of cell types including intestinal epithelial cells. However, the functional responses of intestinal epithelial cells to stimulation with IL-17 are unknown. Moreover, the signal transduction pathways activated by the IL-17R have not been characterized. IL-17 induced NF-kappa B protein-DNA complexes consisting of p65/p50 heterodimers in the rat intestinal epithelial cell line IEC-6. The induction of NF-kappa B correlated with the induction of CXC and CC chemokine mRNA expression in IEC-6 cells. IL-17 acted in a synergistic fashion with IL-1 beta to induce the NF-kappa B site-dependent CINC promoter. Induction of the CINC promoter by IL-17 in IEC-6 cells was TNF receptor-associated factor-6 (TRAF6), but not TRAF2, dependent. Furthermore, IL-17 induction of the CINC promoter could be inhibited by kinase-negative mutants of NF-kappa B-inducing kinase and I kappa B kinase-alpha. In addition to activation of the NF-kappa B, IL-17 regulated the activities of extracellular regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinases in IEC-6 cells. Whereas the IL-17-mediated activation of extracellular regulated kinase mitogen-activated protein kinases was mediated through ras, c-Jun N-terminal kinase activation was dependent on functional TRAF6. These data suggest that NF-kappa B-inducing kinase serves as the common mediator in the NF-kappa B signaling cascades triggered by IL-17, TNF-alpha, and IL-1 beta in intestinal epithelial cells.     

Tyrosine 763 of the murine granulocyte colony-stimulating factor receptor mediates Ras-dependent activation of the JNK/SAPK mitogen-activated protein kinase pathway.

The receptor for granulocyte colony-stimulating factor (G-CSF) can mediate differentiation and proliferation of hemopoietic cells. A proliferative signal is associated with activation of the ERK mitogen-activated protein kinase (MAPK) pathway. To determine whether other MAPK pathways are activated by G-CSF signalling, we have investigated activation of JNK/SAPK in cells proliferating in response to G-CSF. Here we show that G-CSF and interleukin-3 activate JNK/SAPK in two hemopoietic cell lines. The region of the G-CSF receptor required for G-CSF-induced JNK/SAPK activation is located within the C-terminal 68 amino acids of the cytoplasmic domain, which contains Tyr 763. Mutation of Tyr 763 to Phe completely blocks JNK/SAPK activation. However, the C-terminal 68 amino acids are not required for ERK2 activation. We show that activation of JNK/SAPK, like that of ERK2, is dependent on Ras but that higher levels of Ras-GTP are associated with activation of JNK/SAPK than with activation of ERK2. Two separate functional regions of the G-CSF receptor contribute to activation of Ras. The Y763F mutation reduces G-CSF-induced Ras activation from 30 to 35% Ras-GTP to 10 to 13% Ras-GTP. Low levels of Ras activation (10 to 13% Ras-GTP), which are sufficient for ERK2 activation, require only the 100 membrane-proximal amino acids. High levels of Ras-GTP provided by expression of oncogenic Ras are not sufficient to activate JNK/SAPK. An additional signal, also mediated by Tyr 763, is required for activation of JNK/SAPK.     

Oncogenic Ras inhibits anoikis of intestinal epithelial cells by preventing the release of a mitochondrial pro-apoptotic protein Omi/HtrA2 into the cytoplasm

Resistance of cancer cells to anoikis, apoptosis induced by cell detachment from the extracellular matrix, is thought to represent a critical feature of the malignant phenotype. Mechanisms that control anoikis of normal and cancer cells are understood only in part. Previously we found that anoikis of non-malignant intestinal epithelial cells is driven by detachment-induced down-regulation of Bcl-X(L), a protein that blocks apoptosis through preventing the release of death-promoting factors from the mitochondria. Mitochondrial proteins the release of which causes anoikis are presently unknown. Similar to what was previously observed by others for keratinocytes and fibroblasts, we show here that anoikis of intestinal epithelial cells does not involve caspase-9, a target of a mitochondrial protein cytochrome c. Furthermore, Smac/Diablo, another mitochondrial pro-apoptotic factor, does not appear to play a role in detachment-dependent apoptosis of these cells either. Instead, anoikis of intestinal epithelial cells is triggered by the release of a mitochondrial protein Omi/HtrA2, an event driven by detachment-induced down-regulation of Bcl-X(L). Moreover, we established that oncogenic ras inhibits anoikis by preventing the release of Omi/HtrA2. This effect of ras required ras-induced down-regulation of a pro-apoptotic protein Bak and could be blocked by an inhibitor of phosphoinositide 3-kinase, a target of Ras that was previously implicated by us in the down-regulation of Bak and blockade of anoikis. We conclude that Omi/HtrA2 is an inducer of anoikis and an important regulator of ras-induced transformation.     

Juxtacrine activation of epidermal growth factor (EGF) receptor by membrane-anchored heparin-binding EGF-like growth factor protects epithelial cells from anoikis while maintaining an epithelial phenotype

Loss of cell-matrix adhesion is often associated with acute epithelial injury, suggesting that "anoikis" may be an important contributor to cell death. Resistance against anoikis is a key characteristic of transformed cells. When nontransformed epithelia are injured, activation of the epidermal growth factor (EGF) receptor (EGFR) by paracrine/autocrine release of soluble ligands can induce a prosurvival program, but there is generally evidence for concomitant dedifferentiation. The EGFR ligand, heparin-binding EGF-like growth factor (HB-EGF), is synthesized as a membrane-anchored precursor that can activate the EGFR via juxtacrine signaling or can be released and act as a soluble growth factor. In Madin-Darby canine kidney cells, expression of membrane-anchored HB-EGF increases cell-cell and cell-matrix adhesion. Therefore, these studies were designed to test the effects of juxtacrine HB-EGF signaling upon cell survival and epithelial integrity when cells are denied proper cell-matrix interactions. Cells expressing a noncleavable mutated form of membrane-anchored HB-EGF demonstrated increased survival from anoikis, formed larger cell aggregates, and maintained epithelial characteristics even following prolonged detachment from the substratum. Physical association between membrane-anchored HB-EGF and EGFR was observed. Signaling studies indicated synergistic effects of EGFR activation and phosphatidylinositol 3-kinase signaling to regulate apoptotic and survival pathways. In contrast, although administration of exogenous EGF partially suppressed anoikis in wild type cells, it also led to an increased expression of mesenchymal markers, suggesting dedifferentiation. Taken together, we propose a novel role for membrane-anchored HB-EGF in the cytoprotection of epithelial cells.     

Disruption of epithelial cell-matrix interactions induces apoptosis

Cell-matrix interactions have major effects upon phenotypic features such as gene regulation, cytoskeletal structure, differentiation, and aspects of cell growth control. Programmed cell death (apoptosis) is crucial for maintaining appropriate cell number and tissue organization. It was therefore of interest to determine whether cell- matrix interactions affect apoptosis. The present report demonstrates that apoptosis was induced by disruption of the interactions between normal epithelial cells and extracellular matrix. We have termed this phenomenon "anoikis." Overexpression of bcl-2 protected cells against anoikis. Cellular sensitivity to anoikis was apparently regulated: (a) anoikis did not occur in normal fibroblasts; (b) it was abrogated in epithelial cells by transformation with v-Ha-ras, v-src, or treatment with phorbol ester; (c) sensitivity to anoikis was conferred upon HT1080 cells or v-Ha-ras-transformed MDCK cells by reverse- transformation with adenovirus E1a; (d) anoikis in MDCK cells was alleviated by the motility factor, scatter factor. The results suggest that the circumvention of anoikis accompanies the acquisition of anchorage independence or cell motility.     

Differentiated murine F9 teratocarcinoma cells are susceptible to apoptosis upon contact with substrate

Epithelial and endothelial cells are susceptible to a subset of apoptosis known as anoikis. This type of programmed cell death is activated upon disruption of cell-substrate contacts. Here we demonstrate that mouse F9 embryonal carcinoma cell line acquires susceptibility to anoikis upon retinoic acid-induced differentiation towards non-malignant pariental endoderm-like cells. F9 cells survival becomes dependent on the substrate by the 4th day of retinoic acid treatment, when cells assume epithelial phenotype as revealed by actin, alpha-actinin and vinculin expression and distribution, and when focal adhesion contacts are formed. Differentiated F9 cells die in suspension by apoptosis as revealed by oligonucleosomal DNA laddering, DAPI staining and DNA flow cytometry analysis. On the contrary, undifferentiated F9 cells form large multicellular aggregates in suspension and survive. Thus, F9 cell line provides a new model to study pathways involved in both anoikis induction and inhibition.     

Thalidomide inhibits epidermal growth factor-induced cell growth in mouse and human monocytic leukemia cells via Ras inactivation

The effect of thalidomide on epidermal growth factor (EGF)-induced cell growth was examined. Thalidomide inhibited EGF-induced cell growth in mouse and human monocytic leukemia cells, RAW 264.7, U937 and THP-1. Thalidomide inhibited EGF-induced phosphorylation of extracellular signal regulated kinase (ERK) 1/2, but not p38 and stress-activated protein kinase (SAPK)/JNK. The phosphorylation of MEK1/2 and Raf at Ser 338 as the upstream molecules of ERK 1/2 was also prevented by thalidomide. Further, it inhibited EGF-induced Ras activation through preventing the transition to GTP-bound active Ras. Thalidomide inhibited the Ras activation induced by lipopolysaccharide (LPS) and vascular endothelial growth factor (VEGF) as well as EGF. There was no significant difference in the expression and function of EGF receptor between thalidomide-treated and non-treated cells. Therefore, thalidomide was suggested to inhibit EGF-induced cell growth via inactivation of Ras.     

Matrix adhesion and Ras transformation both activate a phosphoinositide 3-OH kinase and protein kinase B/Akt cellular survival pathway.

Upon detachment from the extracellular matrix, epithelial cells enter into programmed cell death, a phenomenon known as anoikis, ensuring that they are unable to survive in an inappropriate location. Activated ras oncogenes protect cells from this form of apoptosis. The nature of the survival signals activated by integrin engagement and usurped by oncogenic Ras are unknown: here we show that in both cases phosphoinositide 3-OH kinase (PI 3-kinase), but not Raf, mediates this protection, acting through protein kinase B/Akt (PKB/Akt). Constitutively activated PI 3-kinase or PKB/Akt block anoikis, while inhibition of PI 3-kinase abrogates protection by Ras, but not PKB/Akt. Inhibition of either PI 3-kinase or PKB/Akt induces apoptosis in adherent epithelial cells. Attachment of cells to matrix leads to rapid elevation of the levels of PI 3-kinase lipid products and PKB/Akt activity, both of which remain high in Ras-transformed cells even in suspension. PI 3-kinase acting through PKB/Akt is therefore implicated as a key mediator of the aberrant survival of Ras-transformed epithelial cells in the absence of attachment, and mediates matrix-induced survival of normal epithelial cells.     

Rapid expression of IL-1beta by intestinal epithelial cells in vitro

Intestinal epithelial cells have been shown to produce IL-1beta in vivo. This gene expression is rapid and precedes most determinants of inflammation, suggesting a pivotal role for IL-1beta in the early events leading to inflammation. To better understand the mechanisms leading to this IL-1beta production, we have developed an in vitro model system employing a nontransformed intestinal epithelial cell line that does not constitutively express IL-1beta. Following detachment, these cells rapidly expressed IL-1beta mRNA. This expression was enhanced, but not induced, by LPS. IL-1beta protein was detected by immunoprecipitation in the culture medium from passaged IEC-18 but not intracellularly, suggesting an efficient secretion of the molecule following induction. Interestingly, culture supernatants from passaged cells were without IL-1 bioactivity, suggesting the presence of an inhibitor as well. RT-PCR and Western blot analysis showed expression of IL-1RII by IEC-18 following detachment, possibly explaining the observed lack of bioactivity. These results indicate a novel pathway for IL-1beta production and suggest that proinflammatory effects of IEC-derived IL-1 may be modulated by the simultaneous production of IL-1 antagonists.     

Protein kinase C signaling mediates a program of cell cycle withdrawal in the intestinal epithelium

Members of the protein kinase C (PKC) family of signal transduction molecules have been widely implicated in regulation of cell growth and differentiation, although the underlying molecular mechanisms involved remain poorly defined. Using combined in vitro and in vivo intestinal epithelial model systems, we demonstrate that PKC signaling can trigger a coordinated program of molecular events leading to cell cycle withdrawal into G(0). PKC activation in the IEC-18 intestinal crypt cell line resulted in rapid downregulation of D-type cyclins and differential induction of p21(waf1/cip1) and p27(kip1), thus targeting all of the major G(1)/S cyclin-dependent kinase complexes. These events were associated with coordinated alterations in expression and phosphorylation of the pocket proteins p107, pRb, and p130 that drive cells to exit the cell cycle into G(0) as indicated by concomitant downregulation of the DNA licensing factor cdc6. Manipulation of PKC isozyme levels in IEC-18 cells demonstrated that PKCalpha alone can trigger hallmark events of cell cycle withdrawal in intestinal epithelial cells. Notably, analysis of the developmental control of cell cycle regulatory molecules along the crypt-villus axis revealed that PKCalpha activation is appropriately positioned within intestinal crypts to trigger this program of cell cycle exit-specific events in situ. Together, these data point to PKCalpha as a key regulator of cell cycle withdrawal in the intestinal epithelium.