β1 integrin/Fak/Src signaling in intestinal epithelial crypt cell survival: integration of complex regulatory mechanisms

The molecular determinants which dictate survival and apoptosis/anoikis in human intestinal crypt cells remain to be fully understood. To this effect, the roles of β1 integrin/Fak/Src signaling to the PI3-K/Akt-1, MEK/Erk, and p38 pathways, were investigated. The regulation of six Bcl-2 homologs (Bcl-2, Mcl-1, Bcl-X_L, Bax, Bak, Bad) was likewise analyzed. We report that: (1) Anoikis causes a down-activation of Fak, Src, Akt-1 and Erk1/2, a loss of Fak–Src association, and a sustained/enhanced activation of p38β, which is required as apoptosis/anoikis driver; (2) PI3-K/Akt-1 up-regulates the expression of Bcl-X_L and Mcl-1, down-regulates Bax and Bak, drives Bad phosphorylation (both serine112/136 residues) and antagonizes p38β activation; (3) MEK/Erk up-regulates Bcl-2, drives Bad phosphorylation (serine112 residue), but does not antagonize p38β activation; (4) PI3-K/Akt-1 is required for survival, whereas MEK/Erk is not; (5) Src acts as a cornerstone in the engagement of both pathways by β1 integrins/Fak, and is crucial for survival; and (6) β1 integrins/Fak and/or Src regulate Bcl-2 homologs as both PI3-K/Atk-1 and MEK/Erk combined. Hence, β1 integrin/Fak/Src signaling translates into integrated mediating functions of p38β activation and regulation of Bcl-2 homologs by PI3-K/Akt-1 and MEK/Erk, consequently determining their requirement (or not) for survival.     

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.     

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.     

Human intestinal epithelial cell survival: differentiation state-specific control mechanisms

To investigate whether human intestinal epithelial cell survival involves distinct control mechanisms depending on the state of differentiation, we analyzed the in vitro effects of insulin, pharmacological inhibitors of Fak, MEK/Erk, and PI3-K/Akt, and integrin (beta1, beta4)-blocking antibodies on the survival of the well-established human Caco-2 enterocyte-like and HIEC-6 cryptlike cell models. In addition, relative expression levels of six Bcl-2 homologs (Bcl-2, Bcl-X(L), Mcl-1, Bax, Bak, and Bad) and activation levels of Fak, Erk-2, and Akt were analyzed. Herein, we report that 1) the enterocytic differentiation process results in the establishment of distinct profiles of Bcl-2 homolog expression levels, as well as p125(Fak), p42(Erk-2), and p57(Akt) activated levels; 2) the inhibition of Fak, of the MEK/Erk pathway, or of PI3-K, have distinct impacts on enterocytic cell survival in undifferentiated (subconfluent Caco-2, confluent HIEC-6) and differentiated (30 days postconfluent Caco-2) cells; 3) exposure to insulin and the inhibition of Fak, MEK, and PI3-K resulted in differentiation state-distinct modulations in the expression of each Bcl-2 homolog analyzed; and 4) Fak, beta1 and beta4 integrins, as well as the MEK/Erk and PI3-K/Akt pathways, are distinctively involved in cell survival depending on the state of cell differentiation. Taken together, these data indicate that human intestinal epithelial cell survival is regulated according to differentiation state-specific control mechanisms.     

Integrin/Fak/Src-mediated regulation of cell survival and anoikis in human intestinal epithelial crypt cells: selective engagement and roles of PI3-K isoform complexes

In human intestinal epithelial crypt (HIEC) cells, the PI3-K/Akt-1 pathway is crucial for the promotion of cell survival and suppression of anoikis. Class I PI3-K consists of a complex formed by a catalytic (C) and regulatory (R) subunit. Three R (p85α, β, and p55γ) and four C (p110α, β, γ and δ) isoforms are known. Herein, we analyzed the expression of PI3-K isoforms in HIEC cells and determined their roles in cell survival, as well as in the β1 integrin/Fak/Src-mediated suppression of anoikis. We report that: (1) the predominant PI3-K complexes expressed by HIEC cells are p110α/p85β and p110α/p55γ; (2) the inhibition and/or siRNA-mediated expression silencing of p110α, but not that of p110β, γ or δ, results in Akt-1 down-activation and consequent apoptosis; (3) the expression silencing of p85β or p55γ, but not that of p85α, likewise induces Akt-1 down-activation and apoptosis; however, the impact of a loss of p55γ on both Akt-1 activation and cell survival is significantly greater than that from the loss of p85β; and (4) both the p110α/p85β and p110α/p55γ complexes are engaged by β1 integrin/Fak/Src signaling; however, the engagement of p110α/p85β is primarily Src-dependent, whereas that of p110α/p55γ is primarily Fak-dependent (but Src-independent). Hence, HIEC cells selectively express PI3-K isoform complexes, translating into distinct roles in Akt-1 activation and cell survival, as well as in a selective engagement by Fak and/or Src within the context of β1 integrin/Fak/Src-mediated suppression of anoikis.     

Human intestinal epithelial cell survival and anoikis. Differentiation state-distinct regulation and roles of protein kinase B/Akt isoforms

We have shown previously that human intestinal epithelial cell survival and anoikis are distinctively regulated according to the state of differentiation. Here we analyzed the roles of protein kinase B/Akt isoforms in such differentiation state distinctions. Anoikis was induced in undifferentiated and differentiated enterocytes by inhibition of focal adhesion kinase (Fak; pharmacologic inhibition or overexpression of dominant-negative mutants) or beta1 integrins (antibody blocking) or by maintaining cells in suspension. Expression/activation parameters of Akt isoforms (Akt-1, Akt-2, and Akt-3) and Fak were analyzed. Activity of Akt isoforms was also blocked by inhibition of phosphatidylinositol 3-kinase or by overexpression of dominant-negative mutants. Here we report the following. 1) The expression/activation levels of Akt-1 increase overall during enterocytic differentiation, and those of Akt-2 decrease, whereas Akt-3 is not expressed. 2) Akt-1 activation is dependent on beta1 integrins/Fak signaling, regardless of the differentiation state. 3) Akt-2 activation is dependent on beta1 integrins/Fak signaling in undifferentiated cells only. 4) Activation of Akt-1 is phosphatidylinositol 3-kinase-dependent, whereas that of Akt-2 is not. 5) Akt-2 does not promote survival or apoptosis/anoikis. 6) Akt-1 is essential for survival. 7) Akt-2 cannot substitute for Akt-1 in the suppression of anoikis. Hence, the expression and regulation of Akt isoforms show differentiation state-specific distinctions that ultimately reflect upon their selective implication in the mediation of human intestinal epithelial cell survival. These data provide new insights into the synchronized regulation of cell survival/death that is required in the dynamic renewal process of tissues such as the intestinal epithelium.     

Merosin-integrin promotion of skeletal myofiber cell survival: Differentiation state-distinct involvement of p60Fyn tyrosine kinase and p38alpha stress-activated MAP kinase

Myofiber survival and suppression of anoikis depend in large part on the merosin (laminin-2/-4)-integrin alpha7beta1D cell adhesion system; however, the question remains as to the nature of the signaling molecules/pathways involved. In the present study, we investigated this question using the C2C12 cell model of myogenic differentiation and its merosin- and laminin-deficient derivatives. Herein, we report that: 1) of four members of the Src family of tyrosine kinases studied (p60Src, p53/56Lyn, p59Yes, or p60Fyn), the expression and activity of p60Fyn are found in myotubes exclusively; 2) a severe decrease of p60Fyn activity correlates with myotube apoptosis/anoikis induced by pharmocological compounds (herbimycin A or PP2) which inhibit tyrosine kinases of the Src family, by merosin deficiency and by beta1 integrin inhibition; 3) myoblast survival depends on Fak and the MEK/Erk pathway, in contrast to myotubes; 4) the PI3-K pathway is not involved in either myoblast or myotube survival; and 5) p38alpha SAPK stimulation and activity (but not that of p38beta) are required in the progression of myotube apoptosis/anoikis induced by p60Fyn inhibition, merosin deficiency or beta1 integrin-inhibition; however, p38 is not involved in myoblast apoptosis. Taken together, these results suggest that the promotion of myotube survival by the merosin-alpha7beta1D adhesion system involves p60Fyn, and that disruptions in this cell adhesion system induce myotube apoptosis/anoikis through a p38alpha SAPK-dependent pathway.     

Src mediates prolactin-dependent proliferation of T47D and MCF7 cells via the activation of focal adhesion kinase/Erk1/2 and phosphatidylinositol 3-kinase pathways

Prolactin (PRL) stimulates breast cancer cell proliferation; however, the involvement of PRL-activated signaling molecules in cell proliferation is not fully established. Here we studied the role of c-Src on PRL-stimulated proliferation of T47D and MCF7 breast cancer cells. We initially observed that PRL-dependent activation of focal adhesion kinase (Fak), Erk1/2, and cell proliferation was mediated by c-Src in T47D cells, because expression of a dominant-negative form of c-Src (SrcDM, K295A/Y527F) blocked the PRL-dependent effects. The Src inhibitor PP1 abrogated PRL-dependent in vivo activation of Fak, Erk1/2, p70S6K, and Akt and the proliferation of T47D and MCF7 cells; Janus kinase 2 (Jak2) activation was not affected. However, in vitro, Fak and Jak2 kinases were not directly inhibited by PP1, demonstrating the effect of PP1 on c-Src kinase as an upstream activator of Fak. Expression of Fak mutant Y397F abrogated PRL-dependent activation of Fak, Erk1/2, and thymidine incorporation, but had no effect on p70S6K and Akt kinases. MAPK kinase 1/2 (Mek1/2) inhibitor PD184352 blocked PRL-induced stimulation of Erk1/2 and cell proliferation; however, p70S6K and Akt activation were unaffected. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 abolished cell proliferation and activation of p70S6K and Akt; however, PRL-dependent activation of Erk1/2 was not modified. Moreover, we show that both c-Src/PI3K and c-Src/Fak/Erk1/2 pathways are involved in the up-regulation of c-myc and cyclin d1 expression mediated by PRL. The previous findings suggest the existence of two PRL-dependent signaling cascades, initiated by the c-Src-mediated activation of Fak/Erk1/2 and PI3K pathways that, subsequently, control the expression of c-Myc and cyclin D1 and the proliferation of T47D and MCF7 breast cancer cells.     

A phosphatidylinositol 3-kinase-dependent pathway that differentially regulates c-Raf and A-Raf

Cytokines trigger the rapid assembly of multimolecular signaling complexes that direct the activation of downstream protein kinase cascades. Two protein kinases that have been linked to growth factor-regulated proliferation and survival are mitogen-activated protein/ERK kinase (MEK) and its downstream target Erk, a member of the mitogen-activated protein kinase family. Using complementary pharmacological and genetic approaches, we demonstrate that MEK and Erk activation requires a phosphatidylinositol 3-kinase (PI3-K)-generated signal in an interleukin (IL)-3-dependent myeloid progenitor cell line. Analysis of the upstream pathway leading to MEK activation revealed that inhibition of PI3-K did not block c-Raf activation, whereas MEK activation was effectively blocked under these conditions. Furthermore, agents that elevated cAMP suppressed IL-3-induced c-Raf activation but did not inhibit MEK activation. Because c-Raf activation and MEK activation were inversely affected by PI3-K- and cAMP-dependent pathways, we examined whether IL-3 activated the alternative Raf isoforms A-Raf and B-Raf. Although IL-3 did not activate B-Raf, A-Raf was activated by the cytokine. Moreover, A-Raf activation, like MEK activation, was blocked by inhibition of PI3-K but was insensitive to cAMP. Experiments with dominant negative mutants of the Raf isoforms showed that overexpression of dominant negative c-Raf did not prevent MEK activation. However, dominant negative A-Raf effectively blocked MEK activation, suggesting that activation of the MEK-Erk signaling cascade is mediated through A-Raf. Taken together, these results suggest that IL-3 receptors engage and activate both c-Raf and A-Raf in hemopoietic cells. However, these intermediates are differentially regulated by upstream signaling cascades and selectively coupled to downstream signaling pathways.     

Differential sensitivity to apoptosis between the human small and large intestinal mucosae: linkage with segment-specific regulation of BCL-2 homologs and involvement of signaling pathways.

The small and large intestines differ in their expression profiles of Bcl-2 homologs. Intestinal segment-specific Bcl-2 homolog expression profiles are acquired as early as by mid-gestation (18-20 weeks) in man. In the present study, we examined the question whether such distinctions underlie segment-specific control mechanisms of intestinal cell survival. Using mid-gestation human jejunum and colon organotypic cultures, we analyzed the impact of growth factors (namely insulin; 10 microg/ml) and pharmacological compounds that inhibit signal transduction molecules/pathways (namely tyrosine kinases, Fak, P13-K/Akt, and MEK/Erk) on cell survival and Bcl-2 homolog expression (anti-apoptotic: Bcl-2, Bcl-X(L), Mcl-1; pro-apoptotic: Bax, Bak, Bad). The relative activation levels of p125Fak, p42Erk-2, and p57Akt were analyzed as well. Herein, we report that (1) the inhibition of signal transduction molecules/pathways revealed striking differences in their impact on cell survival in the jejunum and colon (e.g., the inhibition of p125Fak induced apoptosis with a significantly greater extent in the jejunum [approximately 43%] than in the colon [approximately 24%]); (2) sharp distinctions between the two segments were noted in the modulatory effects of the various treatments on Bcl-2 homolog steady-state levels (e.g., inhibition of tyrosine kinase activities in the jejunum down-regulated all anti-apoptotics analyzed while increasing Bax, whereas the same treatment in the colon down-regulated Bcl-X(L) only and increased all pro-apoptotics); and (3) in addition to their differential impact on cell survival and Bcl-2 homolog expression, the MEK/Erk and P13-K/Akt pathways were found to be distinctively regulated in the jejunum and colon mucosae (e.g., insulin in the jejunum increased p42Erk-2 activation without affecting that of p57Akt, whereas the same treatment in the colon decreased p42Erk-2 activation while increasing that of p57Akt). Altogether, these data show that intestinal cell survival is characterized by segment-specific susceptibilities to apoptosis, which are in turn linked with segmental distinctions in the involvement of signaling pathways and the regulation of Bcl-2 homolog steady-state levels. Therefore, these indicate that cell survival is subject to segment-specific control mechanisms along the proximal-distal axis of the intestine.     

Epidermal growth factor inhibition of c-Myc-mediated apoptosis through Akt and Erk involves Bcl-xL upregulation in mammary epithelial cells

In earlier studies, we and others have established that activation of EGFR can promote survival in association with upregulation of Bcl-x(L). However, the mechanism responsible for upregulation of Bcl-x(L) is unknown. For the current studies we have chosen pro-apoptotic, c-Myc-overexpressing murine mammary epithelial cells (MMECs) derived from MMTV-c-Myc transgenic mouse tumors. We now demonstrate that EGFR activation promotes survival through Akt and Erk1/2. Blockade of EGFR kinase activity and the PI3-K/Akt and MEK/Erk pathways with pharmacological inhibitors resulted in a significant induction of cellular apoptosis, paralleled by a downregulation of both Akt and Erk1/2 proteins. Consistent with a survival-promoting role of Akt, we observed that constitutively activated Akt (Myr-Akt) inhibited apoptosis of pro-apoptotic, c-Myc-overexpressing cells following the inhibition of EGFR tyrosine kinase activity. In addressing possible downstream effectors of EGFR through activated Akt, we detected significant upregulation of Bcl-x(L) protein, suggesting this pro-survival protein is a target of Akt in MMECs. By using pharmacological inhibitors of PI3-K/Akt and MEK/Erk together with dominant-negative Akt and Erk1 we observed the decrease in Bcl-x(L) protein. Our findings may be of importance for understanding the emerging role of Bcl-x(L) as a potential marker of poor prognosis in breast cancer.     

Integrin α8β1 confers anoikis susceptibility to human intestinal epithelial crypt cells

We previously reported that integrin α8β1 is expressed in human intestinal epithelial crypt cells (HIECs) and represents one of the major RGD-binding integrins expressed by these cells. Moreover, the depletion of α8β1 affects vinculin, but not paxillin, localization at focal adhesion points. In the present study, we show that the integrin α8 shRNA-mediated knockdown in HIECs leads to a decrease in anoikis susceptibility under cell suspension culture conditions, marked by a reduction in PARP cleavage and propidium iodide incorporation. Moreover, α8β1-depleted HIECs exhibited an illicitly sustained activation of Fak and PI3-K/Akt-1 under anoikis conditions, rendering them refractory to anoikis. To this effect, colon cancer cells exhibiting resistance to anoikis not only displayed a loss of α8β1 expression, but forced expression of α8β1 in these cells decreased their resistance to anoikis. Consequently, α8β1 is a prerequisite for the proper conduct of anoikis in normal HIECs, whereas its loss contributes to the illicit acquisition of anoikis resistance.     

Dominant roles of the Raf/MEK/ERK pathway in cell cycle progression,prevention of apoptosis and sensitivity to chemotherapeutic drugs

The effects of the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR signaling pathways on cell cycle progression, gene expression, prevention of apoptosis and sensitivity to chemotherapeutic drugs were examined in FL/ΔAkt-1:ER*(Myr⁺) + ΔRaf-1:AR cells which are conditionally-transformed to grow in response to Raf-1 and Akt-1 activation by treatment with testosterone or tamoxifen respectively. In these cells we can compare the effects of normal cytokine vs. oncogene mediated signaling in the same cells by changing the culture conditions. Raf-1 was more effective than Akt-1 in inducing cell cycle progression and preventing apoptosis in the presence and absence of chemotherapeutic drugs. The normal cytokine for these cells, interleukin-3 induced/activated most downstream genes transiently, with the exception of p70S6K that was induced for prolonged periods of time. In contrast, most of the downstream genes induced by either the activate Raf-1 or Akt-1 oncogenes were induced for prolonged periods of time, documenting the differences between cytokine and oncogene mediated gene induction which has important therapeutic consequences. The FL/ΔAkt-1:ER*(Myr⁺) + ΔRaf-1:AR cells were sensitive to MEK and PI3K/mTOR inhibitors. Combining MEK and PI3K/mTOR inhibitors increased the induction of apoptosis. The effects of doxorubicin on the induction of apoptosis could be enhanced with MEK, PI3K and mTOR inhibitors. Targeting the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways may be an effective approach for therapeutic intervention in those cancers which have upstream mutations which result in activation of these pathways.     

The phosphatidylinositol 3-kinase/Akt signaling pathway modulates the endocrine differentiation of trophoblast cells

Activation of Lyn, a Src-related nonreceptor tyrosine kinase, in trophoblast cells is associated with trophoblast giant cell differentiation. The purpose of the present work was to use Lyn as a tool to identify signaling pathways regulating the endocrine differentiation of trophoblast cells. The Src homology 3 domain of Lyn was shown to display differentiation-dependent associations with other regulatory proteins, including phosphatidylinositol 3-kinase (PI3-K). PI3-K activation was dependent upon trophoblast giant cell differentiation. The downstream mediator of PI3-K, Akt/protein kinase B, also exhibited differentiation-dependent activation. Lyn is a potential regulator of the PI3-K/Akt signaling pathway, as are receptor tyrosine kinases. Protein tyrosine kinase profiling was used to identify two candidate regulators of the PI3-K/Akt pathway, fibroblast growth factor receptor-1 and Sky. At least part of the activation of Akt in differentiating trophoblast giant cells involves an autocrine growth arrest-specific-6-Sky signaling pathway. Inhibition of PI3-K activities via treatment with LY294002 disrupted Akt activation and interfered with the endocrine differentiation of trophoblast giant cells. In summary, activation of the PI3-K/Akt signaling pathway regulates the development of the differentiated trophoblast giant cell phenotype.     

Dominant roles of the Raf/MEK/ERK pathway in cell cycle progression,prevention of apoptosis and sensitivity to chemotherapeutic drugs

The effects of the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR signaling pathways on cell cycle progression, gene expression, prevention of apoptosis and sensitivity to chemotherapeutic drugs were examined in FL/ΔAkt-1:ER*(Myr⁺) + ΔRaf-1:AR cells which are conditionally-transformed to grow in response to Raf-1 and Akt-1 activation by treatment with testosterone or tamoxifen respectively. In these cells we can compare the effects of normal cytokine vs. oncogene mediated signaling in the same cells by changing the culture conditions. Raf-1 was more effective than Akt-1 in inducing cell cycle progression and preventing apoptosis in the presence and absence of chemotherapeutic drugs. The normal cytokine for these cells, interleukin-3 induced/activated most downstream genes transiently, with the exception of p70S6K that was induced for prolonged periods of time. In contrast, most of the downstream genes induced by either the activate Raf-1 or Akt-1 oncogenes were induced for prolonged periods of time, documenting the differences between cytokine and oncogene mediated gene induction which has important therapeutic consequences. The FL/ΔAkt-1:ER*(Myr⁺) + ΔRaf-1:AR cells were sensitive to MEK and PI3K/mTOR inhibitors. Combining MEK and PI3K/mTOR inhibitors increased the induction of apoptosis. The effects of doxorubicin on the induction of apoptosis could be enhanced with MEK, PI3K and mTOR inhibitors. Targeting the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways may be an effective approach for therapeutic intervention in those cancers which have upstream mutations which result in activation of these pathways.     

R-Ras promotes focal adhesion formation through focal adhesion kinase and p130(Cas) by a novel mechanism that differs from integrins

R-Ras regulates integrin function, but its effects on integrin signaling pathways have not been well described. We demonstrate that activation of R-Ras promoted focal adhesion formation and altered localization of the alpha2beta1 integrin from cell-cell to cell-matrix adhesions in breast epithelial cells. Constitutively activated R-Ras(38V) dramatically enhanced focal adhesion kinase (FAK) and p130(Cas) phosphorylation upon collagen stimulation or clustering of the alpha2beta1 integrin, even in the absence of increased ligand binding. Signaling events downstream of R-Ras differed from integrins and K-Ras, since pharmacological inhibition of Src or disruption of actin inhibited integrin-mediated FAK and p130(Cas) phosphorylation, focal adhesion formation, and migration in control and K-Ras(12V)-expressing cells but had minimal effect in cells expressing R-Ras(38V). Therefore, signaling from R-Ras to FAK and p130(Cas) has a component that is Src independent and not through classic integrin signaling pathways and a component that is Src dependent. R-Ras effector domain mutants and pharmacological inhibition suggest a partial role for phosphatidylinositol 3-kinase (PI3K), but not Raf, in R-Ras signaling to FAK and p130(Cas). However, PI3K cannot account for the Src-independent pathway, since simultaneous inhibition of both PI3K and Src did not completely block effects of R-Ras on FAK phosphorylation. Our results suggest that R-Ras promotes focal adhesion formation by signaling to FAK and p130(Cas) through a novel mechanism that differs from but synergizes with the alpha2beta1 integrin.     

The tyrosine phosphatase inhibitor orthovanadate mimics NGF-induced neuroprotective signaling in rat hippocampal neurons

Activation of the high affinity neurotrophin receptor tropomyosin-related kinase A (TrkA) by nerve growth factor (NGF) leads to phosphorylation of intracellular tyrosine residues of the receptor with subsequent activation of signaling pathways involved in neuronal survival such as the phosphoinositide-3-kinase (PI3-K)/protein kinase B (PKB/Akt) pathway and the mitogen-activated protein kinase (MAPK) cascade. In the present study, we tested whether inhibition of protein-tyrosine phosphatases (PTP) by orthovanadate could enhance tyrosine phosphorylation of TrkA thereby stimulating NGF-like survival signaling in embryonic hippocampal neurons. We found that the PTP inhibitor orthovanadate (1 microM) enhanced TrkA phosphorylation and protected neurons against staurosporine (STS)-induced apoptosis in a time-and concentration-dependent manner. Inhibition of PTP enhanced TrkA phosphorylation also in the presence of NGF antibodies indicating that NGF binding to TrkA was not required for the effects of orthovanadate. Moreover, orthovanadate enhanced phosphorylation of Akt and the MAPK Erk1/2 suggesting that the signaling pathways involved in the protective effect were similar to those activated by NGF. Accordingly, inhibition of PI3-K by wortmannin and MAPK-kinase (MEK) inhibition by UO126 abolished the neuroprotective effects. In conclusion, the results indicate that orthovanadate mimics the effect of NGF on survival signaling pathways in hippocampal neurons. Thus, PTP inhibition appears to be an appropriate strategy to trigger neuroprotective signaling pathways downstream of neurotrophin receptors.     

Association of PI3K-Akt signaling pathway with digitalis-induced hypertrophy of cardiac myocytes

Our previous studies on cardiac myocytes showed that positive inotropic concentrations of the digitalis drug ouabain activated signaling pathways linked to Na(+)-K(+)-ATPase through Src and epidermal growth factor receptor (EGFR) and led to myocyte hypertrophy. In view of the known involvement of phosphatidylinositol 3-kinase (PI3K)-Akt pathways in cardiac hypertrophy, the aim of the present study was to determine whether these pathways are also linked to cardiac Na(+)-K(+)-ATPase and, if so, to assess their role in ouabain-induced myocyte growth. In a dose- and time-dependent manner, ouabain activated Akt and phosphorylation of its substrates mammalian target of rapamycin and glycogen synthase kinase in neonatal rat cardiac myocytes. Akt activation by ouabain was sensitive to PI3K inhibitors and was also noted in adult myocytes and isolated hearts. Ouabain caused a transient increase of phosphatidylinositol 3,4,5-trisphosphate content of neonatal myocytes, activated class IA, but not class IB, PI3K, and increased coimmunoprecipitation of the alpha-subunit of Na(+)-K(+)-ATPase with the p85 subunit of class IA PI3K. Ouabain-induced activation of ERK1/2 was prevented by Src, EGFR, and MEK inhibitors, but not by PI3K inhibitors. Activation of Akt by ouabain, however, was sensitive to inhibitors of PI3K and Src, but not to inhibitors of EGFR and MEK. Similarly, ouabain-induced myocyte hypertrophy was prevented by PI3K and Src inhibitors, but not by an EGFR inhibitor. These findings 1) establish the linkage of the class IA PI3K-Akt pathway to Na(+)-K(+)-ATPase and the essential role of this linkage to ouabain-induced myocyte hypertrophy and 2) suggest cross talk between these PI3K-Akt pathways and the signaling cascades previously identified to be associated with cardiac Na(+)-K(+)-ATPase.     

Inhibition of integrin-mediated crosstalk with epidermal growth factor receptor/Erk or Src signaling pathways in autophagic prostate epithelial cells induces caspase-independent death

In vivo in the prostate gland, basal epithelial cells adhere to laminin 5 (LM5) via alpha3beta1 and alpha6beta4 integrins. When placed in culture primary prostate basal epithelial cells secrete and adhere to their own LM5-rich matrix. Adhesion to LM5 is required for cell survival that is dependent on integrin-mediated, ligand-independent activation of the epidermal growth factor receptor (EGFR) and the cytoplasmic tyrosine kinase Src, but not PI-3K. Integrin-mediated adhesion via alpha3beta1, but not alpha6beta4 integrin, supports cell survival through EGFR by signaling downstream to Erk. PC3 cells, which do not activate EGFR or Erk on LM5-rich matrices, are not dependent on this pathway for survival. PC3 cells are dependent on PI-3K for survival and undergo caspase-dependent death when PI-3K is inhibited. The death induced by inhibition of EGFR or Src in normal primary prostate cells is not mediated through or dependent on caspase activation, but depends on the induction of reactive oxygen species. In addition the presence of an autophagic pathway, maintained by adhesion to matrix through alpha3beta1 and alpha6beta4, prevents the induction of caspases when EGFR or Src is inhibited. Suppression of autophagy is sufficient to induce caspase activation and apoptosis in LM5-adherent primary prostate epithelial cells.