Nouvelles stratégies anticancéreuses (compréhension moléculaire de l’oncogénèse) / New approaches for cancer treatment based on our molecular understanding of oncogenesis
Colloque organisé par Johannes L. Bos, Pierre Chardin, Alfred Wittinghofer et Frank McCormick du 4 au 9 mai 2004
Allan Balmain, Anton Berns, Katrien Berns, Johannes L. Bos, Pierre Chardin, Jacqueline Cherfils, Hans Clevers, John De Koning, Ludger Johannes, So Young Kim, Alex Levitzki, Daniel Louvard, Richard Marais, Chris Marshall, Madelon Maurice, Frank McCormick, Laurent Meijer, Tony Pawson, Hugues de Thé, Daan Van Aalten, Karen Vousden, Fred Wittinghofer
The workshop « New approaches for cancer treatment based on our molecular understanding of oncogenesis” (May 4-9, 2004) was aimed to bring together scientists that work on various aspects of understanding the mechanism of tumor formation and the translation of this knowledge in the development of new treatments. The Foundation des Treilles gave us the opportunity to organize this meeting at their estate near Nice, France. The excellent surroundings and the exquisite hospitality of the people of the Foundation made this meeting, together with the excellent talks, extensive discussions and a very friendly and collegial atmosphere, unforgettable. The idea for this meeting originated from discussions between a number of the invited speakers about what the next steps will be in translating our molecular understanding of cancer into therapy. In particular, we aimed to discuss (mouse) model systems for better validation of targets to evaluate current “high profile” targets and to discuss new approaches for targeting, including the disruption and/or stabilization of protein-protein interactions. To reach that goal, a number of experts from different areas were invited, which were willing to look beyond what is currently known and to discuss what is required to make further progression in our aims to treat cancer.
Novel approaches to use mice to validate targets and drugs
Anton Berns (Netherlands Cancer Institute, Amsterdam) discussed conditional mouse models for cancer, their utility to mimic human disease and their use for testing therapeutic intervention strategies. He showed that sporadic inactivation of both p53 and Rb in lung by Adenovirus-Cre resulted in the development of Small Cell Lung Cancer. This sporadically induced mouse tumor was indistinguishable from its human counterpart both with respect to phenotypic characteristics and metastatic spread to other tissues. Therefore, this mouse could serve as an important model to gain insight into additional lesions critical in lung cancer and to test new intervention strategies.
He also showed how these sporadic mouse models can be used in drug testing e.g. to determine the best schedule of combination therapies.
A CDK inhibitor can greatly enhance the efficacy of doxorubicin to kill a tumor in which Rb is deleted, but only if the CDK inhibitor was administered before doxorubicin. Administration of the CDK inhibitor itself had no effect. The example showed that the right combination, dose and sequence of administration of drugs could substantially improve the therapeutic outcome. It is evident that mouse models are going to be invaluable to sort out suitable treatment combinations and schedules and in that way will help to accelerate development of new treatment modalities.
Daniel Louvard (Institut Curie, Paris) presented a new transgenic mouse model that expresses oncogenic K-rasV12G under control of the murine villin promoter in intestinal epithelial cells. Over 80% of the transgenic animals displayed single or multiple intestinal lesions, ranging from aberrant crypt foci to invasive adenocarcinomas. Expression of K-rasV12G caused activation of the MAP kinase cascade, and the tumors were frequently characterized by deregulated cellular proliferation. Unexpectedly, no evidence was obtained for inactivating mutations of the tumor suppressor gene Apc, the « gatekeeper » in colonic epithelial proliferation. However, spontaneous mutation of the tumor suppressor gene p53, a frequent feature in the human disease, was found in about 40% of the tumors.This animal model recapitulates the stages of tumor progression as well as genetic alterations found in human colorectal cancer. Furthermore, it demonstrates a role of activated Ras in intestinal tumor progression without the requirement of a previous inactivation of the tumor suppressor Apc. In addition Louvard expressed, in collaboration with the laboratory of Prof. S. Artavanis-Tsakonas (MGH, Harvard), the cytoplasmic domain of Notch under the control of the villin promoter. This constitutively activated truncated receptor stimulates cell proliferation and impairs cell differentiation. The embryonic development of the gut of these transgenic mice is only slightly affected but they are born with a non-functional intestine unable to perform the digestive and barrier functions of a normal intestinal mucosa. This leads to 100% of death three days after birth. The mucosa of new born animal is made of proliferative immature cells, goblet cells and enteroendocrine cells are greatly reduced.
It was suggested that the proliferative cells represent an enhanced population of stem cells and/or its progenitors. This new model should help to characterize and isolate stem cells of the gut.
Allan Balmain (UCSF Cancer Center, San Francisco) discussed the problems associated with the identification of tumor susceptibility genes using both mouse and human systems. Many signaling pathways have overlapping components, and in some cases can have opposing effects on cell growth or apoptosis. Polymorphisms that increase the potency of these signaling molecules therefore have unpredictable effects on susceptibility to cancer development or progression, as the ultimate outcome depends on the genetic background of the host. One way to tackle this problem is to use mouse models to generate large populations for linkage analysis and haplotyping to identify interacting genetic loci that control the cancer phenotype. Network analysis has proven to be a powerful tool for dissection of the genetic basis of complex traits in the mouse and lower eucaryotic systems, and provides a promising approach to analysis of cancer susceptibility in humans. One example was provided of a polymorphic tumor modifier gene identified from mouse crosses that is also polymorphic in humans, and has been associated with increased risk of developing multiple cancer types. This gene – Aurora kinase or STK15 – influences genetic instability by causing chromosome mis-segregation during mitosis, leading to genetic instability. Individuals homozygous for the Ile31 allele have significantly increased risk of cancer development, identifying Aurora-A as a low penetrance tumor susceptibility gene. Identification of potential drug targets for cancer presents a similar set of problems, as many known “cancer genes” such as myc, ras, Cdk6, can have either negative of positive effects on cell growth depending on the context. The use of targeted inhibitors of these pathways will therefore have to be coupled to accurate determination of the specific pathways that are activated in each individual tumor, rather than being used to treat all patients with a particular type of cancer.
Hans Clevers (Hubrecht Laboratory, Utrecht) presented three short stories on the molecular aberrations in human intestinal polyposis syndromes.
Starting off with familial Adenomatous Polyposis, he presented that the mutation of the APC gene in this syndrome leads to the inappropriate activation of the Wnt cascade in the transformed cells. The consequent activation of a Tcf4-driven transcriptional program maintains a progenitor cell phenotype in the mutant cells of the intestinal epithelium.
Reversing the effects on the Wnt pathway in colorectal cancer cells results in G1 arrest and rapid dell differentiation. Using a cell line system and DNA arraying technology, the Tcf4-driven transcriptional target gene program has been partially uncovered. Genes within that program are not only expressed in the adenomas, but also in the crypts of the intestine. Among these genes are the EphB genes, encoding tyrosine kinase receptors involved in cell guidance and cell sorting. Early adenomas as well as crypts express these genes. However, upon progression these genes are switched off. In a transgenic mouse model, it was demonstrated that the secondary silencing of the EphB genes is necessary for tumor progression.
Studying expression and function of BMP signaling in the intestine, Clevers found that stroma within the villus produces BMP4 and thus signals to the epithelium on the villus. This could be inhibited by the transgenic expression of Noggin in the intestine of mice. Unexpectedly, these mice developed a Juvenile Polyposis-like syndrome, which was due to the aberrant development after birth of multiple ectopic crypts. This provides a glimps on the mechanism of hamartoma formation in Juvenile Polyposis, in which many patients carry mutations in components of the BMP pathway, i.e. SMAD4 or BMPRIa.
Finally, Clevers showed recent data implying a role for the intestinal Peutz-Jeghers tumor suppressor gene LKB1 in polarization of epithelial cells in the intestine. LKB1 is the close homolog of polarity genes in worms and flies, i.e. Par4 and dLKB1 respectively. It was found that LKB1 requires association with a pseudokinase STRAD1 to be activated and translocated from the nucleus to the cytoplasm, where it presumably acts. When this system was rebuilt in cell lines using a tetracyclin-inducible system, single cells responded by the formation of polar structures such as a brush border, by the localized positioning of junctional proteins and by the formation of apical and basolateral membrane domains. Likely, loss of the ability to polarize contributes to the initiation of cellular transformation in Peutz-Jeghers hamartomas.
Kinases as molecular targets
Laurant Meyer (CNRS, Roscoff) reported on the quest for pharmacological inhibitors of cyclin dependent kinases (CDKs). Deregulation of CDKs and GSK-3 in various diseases, especially cancers and neurodegenerative disorders, has stimulated an intensive search for selective pharmacological inhibitors.
Some have reached the clinical stage of pharmaceutical evaluation, namely roscovitine (CYC202), currently in phase 2 clinical trials against lung and breast cancers. Over fifty CDK inhibitors and about twenty GSK-3 inhibitors have been identified, among which more than twenty have been co-crystallized with CDK2 and four with GSK-3. These co-crystal structures are extremely helpful to design further derivatives with increased potency and selectivity. These kinase inhibitors all target the ATP-binding pocket of the catalytic site of their targets. The actual selectivity of most compounds, and thus the underlying mechanism of their cellular effects, is poorly known. Affinity chromatography using immobilized inhibitors provides one approach to identify the actual targets of kinase inhibitors. Results show that although some compounds are quite selective, single target products are very unlikely to be discovered. This may in fact be an advantage as multiple targets lower the chance for resistant cells to develop.
Daan van Aalten (Wellcome Trust Biocentre, Dundee) presented the MO25a crystal structure. MO25a is a 40kD protein that together with the STE20-Related ADaptor-a (STRADa) pseudokinase, forms a regulatory complex capable of stimulating the activity of the LKB1 tumour suppressor. MO25a binds directly to a conserved Trp-Glu-Phe sequence at the STRADa C-terminus, markedly enhancing binding of STRADa to LKB1 and increasing LKB1 catalytic activity. The MO25a structure reveals a helical repeat fold, distantly related to the Armadillo proteins. A complex with the STRADa peptide reveals a hydrophobic pocket that is involved in a unique and specific interaction with the Trp-Glu-Phe motif, further supported by mutagenesis studies. Further mutagenesis is beginning to identify residues on the concave surface of the protein that play a role in stabilising the heterotrimeric complex.
The data presented are a first step towards structural analysis of the LKB1-STRAD-MO25 complex, and suggests that MO25a is a scaffold protein to which other regions of STRAD/LKB1, cellular LKB1 substrates or regulatory components could bind.
Richard Marais (Chester Beatty Laboratories, London) discussed whether B-raf is a good target for intervention. B-RAF is mutated in about 70% of melanomas (7% of all cancers) and over 45 mutations have been described. They are mostly in the kinase domain, where there are two clusters, one in the glycine-rich loop and the other in the activation segment. A single activation segment mutant, V599E accounts for about 90% of all B-RAF mutations in cancer. The kinase activity of V599EB-RAF is ~ 500 fold higher than wild-type B-RAF.
Studies were presented using the mouse melanocyte cell line Melan-a. These cells are highly dendritic and make the pigment melanin, but when V599EB-RAF is expressed they loose their dendrites and cease melanin production. They have constitutive ERK activation and their growth is independent of TPA, an essential mitogen for the parental cells. They also grow as colonies in soft agar and as tumours in nude mice. By use of siRNA it was demonstrated that the elevated ERK activity and constitutive DNA synthesis seen in B-RAF transformed melanocytes is dependent on B-RAF and not on C-RAF, whereas ERK activity and DNA synthesis are not affected by either B-RAF or C-RAF depletion in RAS-transformed melanocytes, presumably because there is innate redundancy in the pathway. SiRNA was also used to deplete expression of A-RAF, B-RAF and C-RAF in melanoma lines harbouring oncogenic B-RAF. Depletion of B-RAF blocked constitutive ERK activity in these cells, whereas A-RAF or C-RAF depletion did not affect ERK activity. B-RAF depletion also suppressed DNA synthesis and induced apoptosis in these cells. A-RAF and C-RAF depletion did not affect cell survival. These data validate B-RAF as a therapeutic target in cancer. Importantly the signal transduction inhibitor BAY43-9006 blocked B-RAF activity in vitro.
In addition, melanoma cells BAY43-9006 inhibited ERK activity, blocked proliferation and induced cell death. Finally, BAY43-9006 repressed the growth of human melanoma xenografts in nude mice.
The crystal structure of the B-RAF kinase domain bound to BAY43-9006. This structure revealed that the inhibitor binds to the active cleft, presumably competing for binding of ATP. BAY43-9006 binds to B-RAF through both hydrophobic and electrostatic interactions and provides an explanation for why the compound binds to B-RAF with high affinity. The activation segment and the glycine-rich loop form an atypical interaction that displaces the activation segment, trapping it into a position that is incompatible with kinase activity.
Importantly, many of the amino acids that are responsible for maintaining this interaction are mutated in human cancer and stimulate activation of B-RAF. Furthermore, one of the activation segment phosphorylation sites (T598) is within the interface of this interaction. This provides a model of how B-RAF is activated. Marais proposes that phosphorylation of T598 would disrupt the glycine-rich loop/ activation segment interaction, allowing the activation segment to fold into the active position. Similarly, many of the mutations that occur in cancer also disrupt the interaction that maintains the inactive conformation, allowing the active form to prevail.
Finally, data were presented to show that three of the mutations that occur in human cancer do not have elevated kinase activity, but rather their ability to phosphorylate MEK in vitro is impaired. Surprisingly, these mutants are still able to stimulate ERK signalling in cells because they are able to activate C-RAF, which is then able to signal to MEK and ERK in cells. The mechanism by which the impaired activity B-RAF mutants are able to activate C-RAF is not known.
Frank McCormick (UCSF Cancer Center, San Francisco) reported on the development of a Raf kinase inhibitor, BAY43 9006 that is currently undergoing clinical trials. Raf kinase was chosen as a target in the Ras pathway, after earlier attempts to block Ras directly had failed. Raf is one of several effectors downstream of Ras, but these effectors work synergistically to transform cells. In particular, the PI 3’ kinase pathway collaborates with Raf in cell culture systems, and in human cancers, both pathways are frequently activated.
This is achieved through direct activation of Ras or by a combination of Raf mutation and PTEN loss. BAY 43 9006 is a potent inhibitor of Raf kinase, but also inhibits c-kit, PDGF-R-beta, flt3 and VEGF-Receptor 2. The clinical results on this compound suggest that in renal cell cancer, the primary target is VEGF-Receptor 2, since these tumors are particularly vascularized and depend heavily on VEGF signaling. However, in melanoma, the results are consistent with Raf inhibition. Inhibition of the Raf-MEK-MAPK pathway sensitizes cells to DNA damaging agents in cell cultures, through loss of Mdm2 expression and upregulation of p53. Clinical responses have been observed that may reflect this mechanism, though this has not been proven yet. Dr McCormick stressed that development of this agent took over 10 years, and that the effects are complex.
Development of other targeted therapies is likely to be equally difficult and will require a thorough understanding of signaling pathways in cancer cells and of genome complexity before these therapies will have a major impact on a significant number of major cancers.
Tricking the cancer to commit suicide
Cancer cells develop strong anti-apoptotic signaling pathways and therefore escape many therapeutic regimens. Recognizing this feature of cancer cells, Alex Levitzki (Hebrew University, Jerusalem) has focused on novel approaches aimed at disarming the cancer cell from its anti-apoptotic weaponry and developing novel strategies aimed at enhancing pro-apoptotic signaling pathways selectively in the cancer cell. One of the key elements is to induce in the targeted cancer cells signaling pathways that induce strong by-stander effects, killing neighboring cancer cells that do not express the target, a common situation in the heterogeneous human tumor. For tumors over-expressing the EGF receptors that goal was achieved by targeting them with EGF guided non-viral vectors loaded with double stranded RNA. These dsRNA molecules are internalized by EGF receptor mediated endocytosis and kill only cells the over-express the EGFR leaving cells with normal levels of EGFR intact. Using that reagent he is able to cure mice bearing sizable intracranial human Glioblastma Multiforme.
The efficiency of therapeutic compounds is often limited by restricted access to their intracellular targets.
To develop innovative methods to get around this problem, Ludger Johannes (Institut Curie, Paris) studies the molecular mechanisms of endocytic intracellular distribution pathways, i.e., clathrin-independent endocytosis and retrograde transport. Proteins and lipids that follow the retrograde route avoid recycling to the plasma membrane and degradation in the late endocytic pathway in order to reach other intracellular compartments, such as the Golgi apparatus, the endoplasmic reticulum, and in some instances the cytosol. The non-toxic B-subunit of Shiga toxin is a marker molecule for these routes. Approaches were developed to use the non-toxic B-subunit of Shiga toxin to target diagnostic or therapeutic compounds to Gb3-positive tumor cells. Proof-of-principle was obtained in the villin-RasV12 mouse model of intestinal tumorigenesis in which they could show the in vivo targeting to and specific accumulation of contrast agents for two-photon microscopy in cancer cells.
Fred Wittinghofer (Max Planck Institute, Dortmund) discussed the structure and function of Ras-like GTP binding proteins. The function of these proteins is to switch between the GTP-bound active and the GDP-bound inactive state. They also acts as timers, since the duration of the signal is inversely proportional to the GTPase reaction rate. Therefore the GTPase reaction is of fundamental importance for the function of Ras and other GTP-binding proteins such as Rho, Rap and RheB. Blockage of the GTPase reaction by oncogenic point mutations of Ras leads to tumor formation in many types of human neoplasia. Other diseases such as Neurofibromatosis type I and Tuberous Sclerosis are caused by mutations in or deletion of neurofibromin and tuberin, GAPS that activate the GTPase reaction of Ras and RheB, respectively.
The GTPase of Ras is stimulated about 105 fold by p120-GAP or neurofibromin. The mechanism of this stimulation has been investigated both biochemically and structurally and has shown that GAP actively participates in the GTPase reaction by supplying an arginine into the active site. The positive charge of the arginine side chain contacts the phosphates and stabilizes the transitions state.
It has been shown that the inability of GAP to stimulate hydrolysis in oncogenic Ras is due to the absence of a catalytic residue (Gln61) or due to steric hindrance, but that GTP cleavage in oncogenic Ras can in principle be achieved by small molecules. A similar mechanism accounts also for GAPS of Rho and Rab proteins.
RapGAP and tuberin, the GAP for RheB, have a mechanism of stimulating GTP hydrolysis that is completely different from these GAPs, since they don’t employ an arginine. Instead the incorporation of an asparagine from GAP into the active site of Rap stimulates GTP hydrolysis by 10 fold. Importantly, mutations that in Ras block GTP hydrolysis do not interfere with catalysis. This is a further indication that the GTPase reaction is, at least in principle, a valuable small molecule drug target.
Jacqueline Cherfils (CNRS, Gif-Sur-Yvette) reported on the interfacial inhibition of protein-protein interactions by Brefeldin A. Defective activation of small GTP-binding proteins has been involved in many pathological conditions, including almost every aspect of tumor onset and progression. Guanine nucleotide exchange factors (GEFs), which are crucial in defining the spatio–temporal parameters of, and stimulating the activation of small G proteins by GDP to GTP exchange, are therefore candidate targets for the specific interruption of small G proteins-controlled pathways. Nature has provided us with the first known GEF inhibitor, Brefeldin A (BFA), which blocks the activation of the small G protein Arf1 by the catalytic Sec7 domain of its exchange factors. Crystallographic analysis of an Arf-GDP-BFA-Sec7 complex revealed a remarkable inhibitory mechanism: BFA is buried at the interface between the small G protein and its GEF, hijacking a transient cavity that does not exist in the unbound proteins and disappears at the subsequent step of the exchange reaction. Binding of BFA freezes the small G protein-GEF complex, which cannot undergo the conformational changes that lead to GDP dissociation. Specificity of BFA for certain Arf/Sec7 pairs in the cell is explained by direct and indirect interactions that the drug establishes with both protein partners. Thus, the case of BFA demonstrates that interfacial inhibition is an efficient and selective alternative to competitive inhibition for disrupting protein-protein interactions, even of low affinity.
The crystallographic analysis further suggests that the activation of other families of small G proteins by their GEFs may involve conformational changes and structural characteristics similar to those captured for the Arf/Sec7 system, thus defining small G protein-(GDP)-GEF transient complexes as candidates for interfacial inhibition strategies.
RNA interference (RNAi) has revolutionised functional genomics allowing selective silencing of individual genes through administration or expression of short interfering RNA (siRNA). To make use of this technology for high throughput target identification in mammalian cells, Katrien Berns (Netherlands Cancer Institute, Amsterdam) reported on the development of expression vectors that direct the synthesis of short hairpin RNAs (shRNAs) that act as short interfering RNA (siRNA)-like molecules to stably suppress gene expression. They have constructed a set of retroviral vectors encoding 23, 742 distinct shRNAs, which target 7, 914 different human genes for suppression. This large set of genes has been selected on the association with cancer and other human diseases.
Using this RNA interference library, five novel modulators of the p53 pathway were identified; RPS6KA6 (ribosomal S6 kinase 4, RSK4), HTATIP (histone acetyl transferase TIP60), HDAC4 (histone deacetylase 4), KIAA0828 (a putative S-adenosyl-L-homocysteine hydrolase, SAH3) and CCT2 (T-complex protein 1, b subunit). Suppression of these genes confers resistance to a p53-dependent and p19ARF-dependent proliferation arrest and abolishes a DNA damage-induced G1 cell cycle arrest, raising the possibility that these genes are tumour suppressor genes. The data indicate that the newly-identified genes have a role, either directly or indirectly, in modulating the transcriptional activation of the p53 target gene p21cip1.
A novel strategy, siRNA bar code screens was developed, to rapidly identify individual siRNA vectors associated with a specific phenotype. A potentially useful application of siRNA bar code screens is the identification of synthetic lethal interactions (a combination of two non-lethal mutations that together result in cell death). The identification of such genetic interactions in mammalian cells may facilitate the development of new and more specific classes of anti-cancer drugs.
Karen Vousden (Beatson Institute, Glasgow) considered putative therapeutic targets in the pathways regulating the activity of the p53 tumour suppressor protein. Many cancers show defects in their ability to induce a p53 response, despite retaining a wild type p53 gene, and this is likely to reflect a failure to inactivate HDM2, the protein responsible for inhibiting p53 function in normal cells.
Blocking HDM2 activity is therefore an attractive therapeutic goal, as this would be expected to re-activate p53 and so suppress tumour cell growth. Several mechanisms to inhibit HDM2 were considered, and a screen for small molecule inhibitors of HDM2’s ubiquitin ligase activity was discussed in some detail. Preliminary evidence of the possible efficacy of several promising compounds derived from this screen was described. The potential of the screen to identify inhibitors of other ubiquitin ligases was also explored.
Hugues de Thé (CNRS, Paris) reported on the Modeling of APL pathogenesis through therapeutic response. Previously, they provided a number of findings to explain the sensitivity of the disease to retinoic acid. More recently, they showed that the exquisite sensitivity of APL to arsenic trioxide correlates with the sumolation of the PML/RARA fusion protein, which triggers the degradation of this oncogene. In addition, arsenic syngergizes dramatically with either retinoic acid to trigger terminal differentiation and eradicate APL. These observations were recently extended to patients, providing the first example of therapeutic modeling in genetically engineered mice.
Finally, cAMP also triggers differentiation and apoptosis in APL and surprisingly, can revert RA-resistance in vivo. cAMP directly targets the function of PML/RARA, most likely through a specific phosphorylation of the receptor. In the case of the RA-resistant APL, cAMP restores the ability of the mutant PML/RARA fusion to activate transcription in the presence of RA. Altogether, these findings demonstrate that three clinically relevant therapies all target the PML/RARA oncogene. These therapies are all strongly synergic in vivo, raising the possibility that APL may be cured without using genotoxic agents.
Cell-cell interactions and re-wiring cellular signaling pathways
Many of the cell’s regulatory proteins are constructed in a modular fashion from domains that mediate specific molecular interactions, or possess catalytic activity.
A potential use of this modular polypeptide design is to facilitate evolution. Tony Pawson (Samual Lunenfeld Research Institute, Toronto) has tested this idea in two ways.
In the first approach, they analyzed the ShcA docking protein, which possesses two phosphotyrosine (pTyr) recognition domains (PTB and SH2), and is itself phosphorylated on tyrosine once bound to activated receptor tyrosine kinases (RTK). These ShcA pTyr motifs in turn bind to the SH2 domains of proteins such as Grb2, and can thereby stimulate signaling through the Ras-MAP kinase and PI 3’-kinase pathways. Interestingly, however, these pTyr motifs are rather recent acquisitions of Shc proteins, since they are absent from C. elegans Shc polypeptides, while only one such motif is present in Drosophila Shc. To test the hypothesis that the incorporation of these Tyr-based motifs into Shc has increased not only its connectivity, but also its biological complexity, they have generated an allelelic series of ShcA mutations in the mouse. Embryos homozygous for a null ShcA allele, or for a mutation that inactivates the RTK-binding PTB domain, die from cardiovascular defects. However, mice expressing a ShcA mutant that lacks the ShcA pTyr sites are viable, but suffer from a defect in the maintenance of muscle spindles and the associated monosynaptic stretch reflex circuit. This is apparently due to a requirement for ShcA phosphorylation and signaling in the spindle in response to Neuregulin, which is produced by the sensory axon. This in turn allows for the production of NT-3 by the spindle, which maintains the associated sensory axon. These and other data indicate that Shc proteins are involved in reciprocal cell signaling, and that the acquisition of new physical connections with intracellular signaling pathways increases Shc’s ability to control this complex facet of multicellular organization.
In a second approach to the potential use of modular domains in generating novel signaling pathways, Pawson and co-workers have attempted to artificially couple mitogenic pTyr signaling to an apoptotic cellular response.
To this end, they have fused the SH2 domain of Grb2, or the PTB domain of ShcA, to the death effector domain (DED) of the FADD adaptor protein. FADD is normally involved in coupling receptors such as Fas to initiator Caspases, which upon dimerization are activated and promote apoptosis.
The chimeric DED-SH2 and DED-PTB proteins are able to ectopically recruit Caspase-8 to a transforming variant of the Neu/ErbB2 RTK, and thereby to stimulate Caspase activation and cell death. As a consequence, the adaptors suppress the ability of Neu-transformed cells to form colonies in soft agar, or to form tumours in immunocompromised mice. A similar induction of Caspase-8 activity and cell death was seen upon stimulation of normal cells with high concentrations of EGF. These data argue that interaction domains can be joined in novel combinations to form new signaling pathways. This approach may be of therapeutic benefit, since transformed cells can potentially be killed by rewiring pTyr signaling to the Caspase cell death pathway.
DUBs and cancer
Members of the large family of deubiquitinating enzymes (DUBs) serve as regulator proteins in the ubiquitin system, either by facilitating the proteolytic pathway or by editing the ubiquitination status of substrate proteins, preventing their degradation.
The USP7 protein was reported to control ubiquitination of p53, thereby acting as a tumor suppressor. In order to explore the growth regulatory function of USP7, Madelon Maurice (Hubrecht Laboratory, Utrecht) generated a stable RNAi-inducible LS174T colon carcinoma cell line in which endogenous protein levels of USP7 were knocked down completely 72 hours after induction with doxycycline. LS174T cells were chosen as they have an intact p53 response pathway. Unlike the expected result based on the reported regulation of p53 ubiquitination by USP7, a G1 growth arrest was observed upon RNAi-mediated elimination of the USP7 protein. No alteration in p53 levels was detected, suggesting a p53-independent mechanism of growth arrest induced by elimination of USP7.
To explore the functional consequences of USP7 knockdown, she used this cell line to compare whole cell proteomes on 2D SDS-PAGE before and 48h after knockdown of USP7. At the 48h time-point of RNAi induction, G1 arrest was starting to show but no sign of apoptosis was observed. Alterations in spot intensities of 38 proteins were detected and these proteins were identified by MS/MS analysis. The most striking differences in spot intensity before and after USP7 knockdown were observed for Hp95/Alix, Ku70, Ku80, Hsp75, and glycogen phosphorylase B.
Hp95/Alix, a protein involved in endosomal trafficking, virus budding and apoptosis, was confirmed by immunoblotting to become downregulated upon USP7 removal. The decrease in Hp95/Alix protein levels is not a general consequence of G1 arrest, indicating that this is a direct effect of USP7 removal. Detailed analysis of Ku70, involved in DNA damage repair and transcriptional regulation, revealed an alteration in IEF upon USP7 elimination, possibly due to an altered posttranslational modification.
Overall, USP7 removal by RNAi in LS174T cells affects expression levels and/or posttranslational modification of 38 proteins in LS174T cells. The nature and functional consequences of these protein modifications for cell growth need further exploration.
GTPase signalling pathways as targets to stop invasion
Dysregulation of small GTPase signalling is a common feature of cancer. For example, tumours carrying a mutated oncogenic Ras gene account for about 12-15% of deaths from cancer. While oncogenic mutations of the type that activate Ras have not yet been found in Rho family GTPases, increasing evidence points to overexpression of Rho GTPases in malignant transformation. Over-expression of Rho GTPases appears to be particularly associated with advanced aggressive tumours.
Chris Marshall (Chester Beatty Laboratories, London) investigated the roles of Ras signalling and Rho family GTPase signalling in cancer. Both Ras and Rho signalling pathways are involved in cell motility and invasion. Several transcriptional targets of ERK MAP kinase signalling downstream of Ras, such as CD44, Fra-1, uPAR play significant roles in cell motility and invasion. Signalling downstream of Rho can both promote or inhibit cell motility.
Tumour cells with a spindle morphology that move using Rac dependent lamelliopdia require Rho signalling through Rho-kinase to be down-regulated. This raises the question how the actomyosin contractility needed for retraction of the trailing edge is generated.
They have identified a pathway that acts redundantly with Rho kinase to generate actomyosin contractility for this mode of cell movement. In contrast to cells with a spindle morphology, tumour cells with a rounded morphology have an absolute requirement for Rho kinase for cell movement.
Inhibition of the signalling pathways that are involved in cell motility and invasion is a potential therapeutic avenue to prevent metastasis. Their work and that of others suggests that Rho kinase may be a good target for anti-invasion therapies. However it is now clear that tumour cells can use different modes of movement depending on the conditions they experience. Thus inhibition of extra-cellular proteolysis results in cells switching from an elongated mesenchymal movement to a rounded form of movement. Interestingly because this rounded form of movement is absolutely dependent on Rho kinase activity, the combination of protease inhibition and Rho kinase inhibition is effective in blocking tumour cell invasion.
Because of the plasticity of the invasive phenotype, they believe that effective regimes to block invasion will have to target multiple signalling events. Significantly blocking the motility of endothelial cells in tumour angiogenesis has also been shown to require combined inhibition of extra-cellular proteolysis and Rho kinase suggesting that the plasticity of the invasive programme is not just a property of tumour cells.
Johannes Bos (UMCU, Utrecht) reported further on the crucial role of Rap1 in inside-out signalling to integrins: Rap1 is both necessary and sufficient to induce a4b1-integrin-mediated cell adhesion. This increased adhesion process is most likely due to an increased clustering (avidity) of integrins. In addition, evidence was presented that Rap1 is also involved in E-cadherin-mediated signalling. When Rap1 was inhibited, MDCK cells fail to form homodimers with E-cadherin coupled to plastic. Furthermore, activation of Rap1 inhibits HGF-induced scattering of MDCK cells, supporting a role for Rap1 in the maintenance of cell-cell junctions.
John de Koning (UMC Utrecht) reported on the crystal structure of the regulatory region of Epac2, which revealed that the highly conserved glutamate residue, present in all known cAMP-binding domains and forming hydrogen bonds with the 2’-hydroxyl of the cAMP ribose group, is absent in the cAMP-binding domain of Epac1 and Epac2. Based on this structure, a large number of cAMP analogs were synthesized lacking the 2’-hydroxyl group, which is absolutely essential for efficient binding and activation of PKA. Several of these, in particular 8-pCPT-2’-O-Me-cAMP, were shown to be very efficient activators of Epac, both in vitro and in vivo, without having an effect neither on PKA activity nor on cAMP-dependent ion-channels.
Using these specific Epac activators, it was shown that Epac is involved in cAMP-regulated cell adhesion, cell junction formation as well as in cAMP-mediated modulation of insulin secretion by pancreatic beta cells. It is anticipated that this list of functions will grow. It thus seems that Epac may be a suitable drug target for cancer as well as diabetes.
Mammalian Target of Rapamycin (mTOR) is a Ser/Thr kinase that plays a key role in the regulation of cell growth and proliferation primarily through control of the cellular translational capacity. mTOR activity is modulated by several independent inputs, including growth factor signaling, amino acid sufficiency and cellular energy levels. Proper regulation of mTOR signaling is frequently lost during tumorigenesis due to genetic alterations that lead to increased PI3-kinase signaling, prominently, loss of the PTEN tumor suppressor. Previous work suggested that mTOR can function as an energy sensor by responding to ATP levels and modulating its activity accordingly. So Young Kim (Friedrich Miescher Institute, Basel) now reported that this effect may be mediated by the AMP-activated kinase (AMPK), which is activated in response to increases in the AMP:ATP ratio. There appears to be a very tight correlation between AMPK activation and inhibition of mTOR signaling in response to various ATP depleting treatments. Furthermore, activation of AMPK, either chemically or through use of constitutive active AMPK mutants, is sufficient to inhibit stimulation of mTOR in response to amino acids and, to a lesser degree, to insulin.
Contradictory to previously published data, it was demonstrated that the ability of AMPK or energy depletion to regulate mTOR signaling is independent of the Tuberous Sclerosis complex (TSC); however, it is unclear what role the small GTPase regulated by TSC, Rheb, plays in this process. Finally, energy depletion and amino acid deprivation both alter the conformation of the mTOR complex, rendering it non-permissive for signaling to downstream substrates.