Incomplete inhibition of phosphorylation of 4E-BP1 as a mechanism of primary resistance to ATP-competitive mTOR inhibitors.

The mammalian target of rapamycin (mTOR) regulates cell growth by integrating nutrient and growth factor signaling and is strongly implicated in cancer. But mTOR is not an oncogene, and which tumors will be resistant or sensitive to new adenosine triphosphate (ATP) competitive mTOR inhibitors now in clinical trials remains unknown. We screened a panel of over 600 human cancer cell lines to identify markers of resistance and sensitivity to the mTOR inhibitor PP242. RAS and phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) mutations were the most significant genetic markers for resistance and sensitivity to PP242, respectively; colon origin was the most significant marker for resistance based on tissue type. Among colon cancer cell lines, those with KRAS mutations were most resistant to PP242, whereas those without KRAS mutations most sensitive. Surprisingly, cell lines with co-mutation of PIK3CA and KRAS had intermediate sensitivity. Immunoblot analysis of the signaling targets downstream of mTOR revealed that the degree of cellular growth inhibition induced by PP242 was correlated with inhibition of phosphorylation of the translational repressor eIF4E-binding protein 1 (4E-BP1), but not ribosomal protein S6 (rpS6). In a tumor growth inhibition trial of PP242 in patient-derived colon cancer xenografts, resistance to PP242-induced inhibition of 4E-BP1 phosphorylation and xenograft growth was again observed in KRAS mutant tumors without PIK3CA co-mutation, compared with KRAS wild-type controls. We show that, in the absence of PIK3CA co-mutation, KRAS mutations are associated with resistance to PP242 and that this is specifically linked to changes in the level of phosphorylation of 4E-BP1.

P190A RhoGAP is required for mammary gland development.

P190A and p190B Rho GTPase activating proteins (GAPs) are essential genes that have distinct, but overlapping roles in the developing nervous system. Previous studies from our laboratory demonstrated that p190B is required for mammary gland morphogenesis, and we hypothesized that p190A might have a distinct role in the developing mammary gland. To test this hypothesis, we examined mammary gland development in p190A-deficient mice. P190A expression was detected by in situ hybridization in the developing E14.5day embryonic mammary bud and within the ducts, terminal end buds (TEBs), and surrounding stroma of the developing virgin mammary gland. In contrast to previous results with p190B, examination of p190A heterozygous mammary glands demonstrated that p190A deficiency disrupted TEB morphology, but did not significantly delay ductal outgrowth indicating haploinsufficiency for TEB development. To examine the effects of homozygous deletion of p190A, embryonic mammary buds were rescued by transplantation into the cleared fat pads of SCID/Beige mice. Complete loss of p190A function inhibited ductal outgrowth in comparison to wildtype transplants (51% vs. 94% fat pad filled). In addition, the transplantation take rate of p190A deficient whole gland transplants from E18.5 embryos was significantly reduced compared to wildtype transplants (31% vs. 90%, respectively). These results suggest that p190A function in both the epithelium and stroma is required for mammary gland development. Immunostaining for p63 demonstrated that the myoepithelial cell layer is disrupted in the p190A deficient glands, which may result from the defective cell adhesion between the cap and body cell layers detected in the TEBs. The number of estrogen- and progesterone receptor-positive cells, as well as the expression levels of these receptors was increased in p190A deficient outgrowths. These data suggest that p190A is required in both the epithelial and stromal compartments for ductal outgrowth and that it may play a role in mammary epithelial cell differentiation.

EMT, cancer stem cells and drug resistance: an emerging axis of evil in the war on cancer.

Tumors are cellularly and molecularly heterogeneous, with subsets of undifferentiated cancer cells exhibiting stem cell-like features (CSCs). Epithelial to mesenchymal transitions (EMT) are transdifferentiation programs that are required for tissue morphogenesis during embryonic development. The EMT process can be regulated by a diverse array of cytokines and growth factors, such as transforming growth factor (TGF)-beta, whose activities are dysregulated during malignant tumor progression. Thus, EMT induction in cancer cells results in the acquisition of invasive and metastatic properties. Recent reports indicate that the emergence of CSCs occurs in part as a result of EMT, for example, through cues from tumor stromal components. Recent evidence now indicates that EMT of tumor cells not only causes increased metastasis, but also contributes to drug resistance. In this review, we will provide potential mechanistic explanations for the association between EMT induction and the emergence of CSCs. We will also highlight recent studies implicating the function of TGF-beta-regulated noncoding RNAs in driving EMT and promoting CSC self-renewal. Finally we will discuss how EMT and CSCs may contribute to drug resistance, as well as therapeutic strategies to overcome this clinically.

Molecular targeted therapy of lung cancer: EGFR mutations and response to EGFR inhibitors.

Somatic mutations within the kinase domain of the epidermal growth factor receptor (EGFR) are present in approximately 10% of non-small-cell lung cancer (NSCLC), with an increased frequency in adenocarcinomas arising in nonsmokers, women, and individuals of Asian ethnicity. These mutations lead to altered downstream signaling by the receptor and appear to define a subset of NSCLC characterized by "oncogene addiction" to the EGFR pathway, which displays dramatic responses to the reversible tyrosine kinase inhibitors gefitinib and erlotinib. The rapid acquisition of drug resistance in most cases, either through mutation of the "gateway" residue in the EGFR kinase domain or by alternative mechanisms, appears to limit the impact on patient survival. Irreversible inhibitors of EGFR display continued effectiveness in vitro against cells with acquired resistance and are now undergoing genotype-directed clinical trials. The molecular and clinical insights derived from targeting EGFR in NSCLC offer important lessons for the broader application of targeted therapeutic agents in solid tumors.

Rac 'n Rho: the music that shapes a developing embryo.

The small GTPases of the Rho subfamily constitute a group of evolutionarily conserved proteins that mediate signaling pathways that regulate a variety of cellular processes, many of which are associated with dynamic cytoskeletal reorganization. These processes determine the shape, adhesive properties, and movement of cells, and the Rho GTPases have therefore been implicated in the complex morphogenesis of tissues in developing multicellular organisms. The Drosophila genetic system has proved particularly useful in establishing the in vivo functions of several of the Rho GTPases and their associated signaling pathway components during development.

Role of prenylation in the interaction of Rho-family small GTPases with GTPase activating proteins.

The role of prenylation in the interaction of Rho-family small GTPases with their GTPase activating proteins (GAPs) was investigated. Prenylated and nonprenylated small GTPases were expressed in Sf9 insect cells and Escherichia coli, respectively. Nucleotide binding to and hydrolysis by prenylated and nonprenylated proteins were identical, but three major differences were observed in their reactions with GAPs. (1) Membrane-associated GAPs accelerate GTP hydrolysis only on prenylated Rac1 and RhoA, but they are inactive on the nonprenylated form of these proteins. The difference is independent of the presence of detergents. In contrast to Rac1 and RhoA, nonprenylated Cdc42 is able to interact with membrane-localized GAPs. (2) Full-length p50RhoGAP and p190RhoGAP react less intensely with nonprenylated Rac1 than with the prenylated protein, whereas no difference was observed in the reaction of isolated GAP domains of either p50RhoGAP or Bcr with the different types of Rac1. (3) Fluoride exerts a significant inhibitory effect only on the interaction of prenylated Rac1 with the isolated GAP domains of p50RhoGAP or Bcr. The effect of fluoride is not influenced by addition or chelation of Al(3+). This is the first detailed study demonstrating that prenylation of the small GTPase is an important factor in determining its reaction with GAPs. It is suggested that both intramolecular interactions and membrane targeting of GAP proteins represent potential mechanisms regulating Rac signaling.

p190 RhoGAP is the principal Src substrate in brain and regulates axon outgrowth, guidance and fasciculation.

The Src tyrosine kinases have been implicated in several aspects of neural development and nervous system function; however, their relevant substrates in brain and their mechanism of action in neurons remain to be established clearly. Here we identify the potent Rho regulatory protein, p190 RhoGAP (GTPase-activating protein), as the principal Src substrate detected in the developing and mature nervous system. We also find that mice lacking functional p190 RhoGAP exhibit defects in axon guidance and fasciculation. p190 RhoGAP is co-enriched with F-actin in the distal tips of axons, and overexpressing p190 RhoGAP in neuroblastoma cells promotes extensive neurite outgrowth, indicating that p190 RhoGAP may be an important regulator of Rho-mediated actin reorganization in neuronal growth cones. p190 RhoGAP transduces signals downstream of cell-surface adhesion molecules, and we find that p190-RhoGAP-mediated neurite outgrowth is promoted by the extracellular matrix protein laminin. Together with the fact that mice lacking neural adhesion molecules or Src kinases also exhibit defects in axon outgrowth, guidance and fasciculation, our results suggest that p190 RhoGAP mediates a Src-dependent adhesion signal for neuritogenesis to the actin cytoskeleton through the Rho GTPase.

The adhesion signaling molecule p190 RhoGAP is required for morphogenetic processes in neural development.

Rho GTPases direct actin rearrangements in response to a variety of extracellular signals. P190 RhoGAP (GTPase activating protein) is a potent Rho regulator that mediates integrin-dependent adhesion signaling in cultured cells. We have determined that p190 RhoGAP is specifically expressed at high levels throughout the developing nervous system. Mice lacking functional p190 RhoGAP exhibit several defects in neural development that are reminiscent of those described in mice lacking certain mediators of neural cell adhesion. The defects reflect aberrant tissue morphogenesis and include abnormalities in forebrain hemisphere fusion, ventricle shape, optic cup formation, neural tube closure, and layering of the cerebral cortex. In cells of the neural tube floor plate of p190 RhoGAP mutant mice, polymerized actin accumulates excessively, suggesting a role for p190 RhoGAP in the regulation of +Rho-mediated actin assembly within the neuroepithelium. Significantly, several of the observed tissue fusion defects seen in the mutant mice are also found in mice lacking MARCKS, the major substrate of protein kinase C (PKC), and we have found that p190 RhoGAP is also a PKC substrate in vivo. Upon either direct activation of PKC or in response to integrin engagement, p190 RhoGAP is rapidly translocated to regions of membrane ruffling, where it colocalizes with polymerized actin. Together, these results suggest that upon activation of neural adhesion molecules, the action of PKC and p190 RhoGAP leads to a modulation of Rho GTPase activity to direct several actin-dependent morphogenetic processes required for normal neural development.

Rho family GTPases: more than simple switches.

Rho family GTPases control a large variety of biological processes. Cycling of Rho proteins between the GDP-bound and the GTP-bound state is controlled by several classes of regulatory proteins. In this review, we discuss the signal-transduction mechanisms that control these regulators. We will emphasize the subcellular localization of Rho GTPases and their regulatory proteins and the role of GTP hydrolysis in signal transmission.

MEK kinase 2 binds and activates protein kinase C-related kinase 2. Bifurcation of kinase regulatory pathways at the level of an MAPK kinase kinase.

MEK kinase 2 (MEKK2) is a 70-kDa protein serine/threonine kinase that has been shown to function as a mitogen-activated protein kinase (MAPK) kinase kinase. MEKK2 has its kinase domain in the COOH-terminal moiety of the protein. The NH(2)-terminal moiety of MEKK2 has no signature motif that would suggest a defined regulatory function. Yeast two-hybrid screening was performed to identify proteins that bind MEKK2. Protein kinase C-related kinase 2 (PRK2) was found to bind MEKK2; PRK2 has been previously shown to bind RhoA and the Src homology 3 domain of Nck. PRK2 did not bind MEKK3, which is closely related to MEKK2. The MEKK2 binding site maps to amino acids 637-660 in PRK2, which is distinct from the binding sites for RhoA and Nck. This sequence is divergent in the closely related kinase PRK1, which did not bind MEKK2. In cells, MEKK2 and PRK2 are co-immunoprecipitated and PRK2 is activated by MEKK2. Similarly, purified recombinant MEKK2 activated PRK2 in vitro. MEKK2 activation of PRK2 is independent of MEKK2 regulation of the c-Jun NH(2)-terminal kinase pathway. MEKK2 activation of PRK2 results in a bifurcation of signaling for the dual control of MAPK pathways and PRK2 regulated responses.

RAFTK/Pyk2 tyrosine kinase mediates the association of p190 RhoGAP with RasGAP and is involved in breast cancer cell invasion.

Focal adhesions and actin cytoskeleton are involved in cell growth, shape and movement and in tumor invasion. Mitogen-induced changes in actin cytoskeleton are accompanied by changes in the tyrosine phosphorylation of several focal adhesion proteins. In this study, we have investigated the role of RAFTK, a cytoplasmic tyrosine kinase related to focal adhesion kinase (FAK), in heregulin-mediated signal transduction in breast cancer cells. Stimulation of T47D cells with heregulin (HRG) induced the tyrosine phosphorylation of RAFTK and the formation of a multiprotein complex. Analyses of the members of the HRG-stimulated complex revealed that RAFTK is associated with p190 RhoGAP (p190), RasGAP and ErbB-2, and plays an essential role in mediating the tyrosine phosphorylation of p190 by Src. Mutation of the Src binding site within RAFTK (402) abolished the phosphorylation of p190. In addition, upon HRG stimulation of T47D cells, association of ErbB-2 with RAFTK was observed and found to be indirect and mediated by Src. Expression of wild-type RAFTK (WT) significantly increased MDA-MB-435 and MCF-7 breast cancer cell invasion, while expression of the kinase-mutated RAFTK-R457 (KM) or the Src binding site mutant RAFTK (402) did not affect this cell invasion. Furthermore, HRG leads to the activation of MAP kinase which is mediated by RAFTK. These findings indicate that RAFTK serves as a mediator and an integration point between the GAP proteins and HRG-mediated signaling in breast cancer cells, and implicate RAFTK involvement in the MAP kinase pathway and in breast cancer cell invasion.

The role of rho family GTPases in development: lessons from Drosophila melanogaster.

It has become increasingly clear in the last few years that the Rho family GTPases regulate cytoskeleton rearrangements that are essential for a variety of morphogenetic events associated with the development of multicellular organisms. In particular, Drosophila has provided an excellent in vivo system for deciphering the signaling pathways mediated by Rho GTPases, as well as establishing the role of these pathways in numerous developmental processes. Continued use of this system will undoubtedly lead to the identification of additional Rho signalling components and information regarding the function and organization of the Rho signaling pathways in tissue morphogenesis. The striking similarity between Drosophila and mammalian Rho signaling components identified thus far indicates that the Rho pathways are highly conserved in evolution. Therefore, the findings from the Drosophila system can be extrapolated to higher organisms, including humans. Combined with the rapid progress in the human and Drosophila genome projects, these findings should contribute greatly to our understanding of mammalian Rho GTPase signaling pathways and their roles in normal development and pathological conditions.

Inhibition of RhoGAP activity is sufficient for the induction of Rho-mediated actin reorganization.

It is generally believed that the induction of actin cytoskeleton rearrangements by extracellular stimuli results from the activation of guanine nucleotide exchange factors for the Rho GTPases. Here, we present evidence that the inactivation of RhoGAP (GTPase activating protein) activity is an equally effective means of promoting Rho-mediated cellular processes. We observed that exposure of cultured fibroblasts to sodium fluoride (NaF) results in a rapid and potent stimulation of actin stress fiber formation. This effect is mediated by the Rho GTPase and is associated with the inactivation of cellular RhoGAP activity. Specifically, NaF promotes formation of a high-affinity complex between Rho and the two cellular p190 RhoGAPs in vivo, apparently sequestering limiting amounts of RhoGAP activity, thereby resulting in Rho activation. p190 RhoGAP activity was found to account for approximately 60% of the total RhoGAP activity detected in whole cell extracts, indicating that relatively small changes in cellular RhoGAP activity can have potent effects on Rho activation. We also found that sub-effective concentrations of NaF combined with sub-effective concentrations of the Rho pathway activator, lysophosphatidic acid, which stimulates guanine nucleotide exchange activity on the Rho GTPase, results in the rapid induction of actin stress fibers. Together, these results suggest that the Rho GTPase is regulated by a fine balance of nucleotide exchange and RhoGAP activities, and that inactivation of RhoGAP activity may be a physiologically important regulatory mechanism for activating the Rho GTPase.

The Drosophila Pkn protein kinase is a Rho/Rac effector target required for dorsal closure during embryogenesis.

The PKN family of PKC-related protein kinases constitutes the major Rho GTPase-associated protein kinase activities detected in mammalian tissues. However, the biological functions of these kinases are unknown. We have identified a closely related PKN homolog in Drosophila (Pkn) that binds specifically to GTP-activated Rho1 and Rac1 GTPases through distinct binding sites on Pkn. The interaction of Pkn with either of these GTPases results in increased kinase activity, suggesting that Pkn is a shared Rho/Rac effector target. Characterization of a loss-of-function mutant of Drosophila Pkn revealed that this kinase is required specifically for the epidermal cell shape changes during the morphogenetic process of dorsal closure of the developing embryo. Moreover, Pkn, as well as the Rho1 GTPase, mediate a pathway for cell shape changes in dorsal closure that is independent of the previously reported Rac GTPase-mediated Jun amino (N)-terminal kinase (JNK) cascade that regulates gene expression required for dorsal closure. Thus, it appears that distinct but coordinated Rho- and Rac-mediated signaling pathways regulate the cell shape changes required for dorsal closure and that Pkn provides a GTPase effector function for cell shape changes in vivo, which acts together with a Rac-JNK transcriptional pathway in the morphogenesis of the Drosophila embryo.

Rho GTPases in development.

It is becoming increasingly clear that the complex family of Rho-related GTPases and their associated regulators and targets are essential mediators of a variety of morphogenetic events required for normal development of multicellular organisms. It is worth noting that the results obtained thus far indicate that the Rho family proteins are largely associated with the regulation of morphogenesis, as opposed to other essential developmental processes such as cell proliferation and cell fate determination. Accumulating evidence also suggests that the role of these proteins and their associated signaling pathways in morphogenesis is in many, but not necessarily all, cases related to their ability to affect the organization of the actin cytoskeleton. Thus, these in vivo observations have served to corroborate similar findings in numerous cultured cell studies. As described, the power of genetics, particularly in Drosophila and C. elegans, has been critical to the recent identification and functional characterization of several Rho family signaling components. Moreover, evidence suggests that the highly evolutionarily conserved structures of many of these proteins translate into conservation of function as well. Thus, it will be possible, in many cases, to extrapolate the findings in the simple systems described herein to higher eukaryotes, including humans. Expanding use of these genetic model systems to dissect Rho-mediated signaling pathways in vivo will undoubtedly lead to a flood of new insights into the organization and function of these pathways in the coming years, especially in development. As the C. elegans genome sequencing effort nears completion and with the Drosophila genome project well underway, the identification of novel relevant genes will proceed with even greater speed. In addition, the rapidly expanding use of mouse knockout strategies, combined with recent developments in the associated knockout technology, will also contribute greatly to the investigation of mammalain Rho signaling pathways and their roles in development.

Myoblast city, the Drosophila homolog of DOCK180/CED-5, is required in a Rac signaling pathway utilized for multiple developmental processes.

The Rac and Cdc42 GTPases share several regulators and effectors, yet perform distinct biological functions. The factors determining such specificity in vivo have not been identified. In a mutational screen in Drosophila to identify Rac-specific signaling components, we isolated 11 alleles of myoblast city (mbc). mbc mutant embryos exhibit defects in dorsal closure, myogenesis, and neural development. DOCK180, the mammalian homolog of Mbc, associates with Rac, but not Cdc42, in a nucleotide-independent manner. These results suggest that Mbc is a specific upstream regulator of Rac activity that mediates several morphogenetic processes in Drosophila embryogenesis.

Activation of the c-fos serum response element by phosphatidyl inositol 3-kinase and rho pathways in HeLa cells.

Many growth factors rapidly induce transcription of the c-fos proto-oncogene. We have investigated the pathways for induction of the c-fos promoter by serum and epidermal growth factor (EGF) in HeLa cells. Induction of the serum response element (SRE) of the c-fos promoter could be split into two parts, one involving the serum response factor-associated ternary complex factor (TCF) factors and the second mediated by core SRE sequences. Serum induction was mediated primarily by the core SRE, whereas EGF used both the TCF and core SRE pathways. Using activated and inhibitory signaling proteins, we found that phosphatidyl inositol 3-kinase (PI3K) and rho family members could mediate activation by serum. Activation by PI3K was mediated by core SRE sequences and was dependent upon rac and rho, suggesting a PI3K-to-rac-to-rho pathway for core SRE activation. The PI3K target Akt was also capable of activating the SRE but functioned through the TCF pathway, suggesting that Akt does not mediate the primary PI3K pathway to the SRE and that Akt is capable of activating TCF family members. Serum and EGF induction of the core SRE was partially inhibited by rho and PI3K inhibitors. The use of these inhibitors demonstrates the complexity of signaling pathways to the SRE and suggests that serum activates rho by PI3K-dependent and -independent pathways.

E1B 55K sequesters WT1 along with p53 within a cytoplasmic body in adenovirus-transformed kidney cells.

WT1 encodes a tumor suppressor that is expressed in cells of the developing kidney and is inactivated in Wilms tumor, a pediatric kidney cancer. The adenovirus E1B 55K gene product contributes to the transformation of primary baby rat kidney (BRK) cells by binding and inactivating the product of the p53 tumor suppressor. We have previously demonstrated that WT1 and p53 are present within a protein complex in vivo. We now show that WT1 is physically associated with E1B 55K in adenovirus-transformed cells, an interaction that is mediated by the first two zinc fingers of WT1. Immunodepletion of p53 abrogates the coimmunoprecipitation of E1B 55K and WT1, consistent with the presence of a trimeric protein complex containing these three proteins. In the presence of E1B 55K, WT1 which is normally localized in the nucleus, is retained within a very high molecular weight complex and sequestered in the characteristic perinuclear cytoplasmic body that contains E1B 55K and p53. Expression of E1B 55K in osteosarcoma cells that undergo apoptosis following expression of WT1 inhibits WT1-mediated cell death. We conclude that E1B 55K may target WT1 along with p53, resulting in the functional inactivation of both tumor suppressor gene products by this viral oncoprotein.