In general, we can differentiate cellular responses to physical force into a purely mechanical response predominantly consisting of the cell's load-bearing deformation of cytoskeletal structures [ 3 ], and into biochemical signaling cascades where force propagation is relayed through membrane proteins or protein complexes to intracellular chemical signaling networks.
Alterations in mechanotransduction often result in diseases such as cancer [ 4 ], arthritis [ 5 ] or atherosclerosis [ 6 ].
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Resolving the mechanisms underlying mechanochemical coupling is therefore of fundamental importance. One emerging mechanism through which mechanical forces may affect downstream signal transduction pathways involves the spatial organization of cell surface receptors [ 7 ]. A special case is that of juxtacrine interactions; for example, ephrin-A1 on one cell binds to EphA2 receptor tyrosine kinase on the apposed cell surface, which will induce assembly of higher-order clusters that trigger bidirectional signaling cascades in interacting cells [ 8 , 9 ].
Recent advances using nanolithography provide new insights into how the ephrin-Eph signaling system responds to different mechanical aspects of interacting cells [ 11 ]. These findings represent an important step towards understanding mechanochemical coupling and give us a glimpse into the significance of mechanical force in health and disease.
In their recent study, Salaita and colleagues have established a procedure for investigating spatiomechanical concepts involved in the EphA2 signaling pathway [ 11 ]. The authors managed to reconstitute in vitro the juxtacrine signaling geometry between living cells expressing the EphA2 receptor tyrosine kinase and the laterally mobile ephrin-A1 ligand displayed on a fluid lipid bilayer supported on a glass substrate. Furthermore, by employing nanolithography the researchers were able to set physical barriers to the ligand mobility on the supported membrane.
Their work shows that the mechanical ligand restriction extends to the spatial organization of EphA2 receptor at cell surface junctions and alters the cellular response to ephrin-A1. Salaita and coworkers scrutinized two experimental conditions: one in which EphA2-expressing cells are interacting with ephrin-A1 ligand that has an unrestricted lateral mobility on a fully saturated lipid bilayer, and a second where ephrin is presented on a fluid membrane that is physically constrained by an underlying pattern of nanofabricated metal lines.
In the first scenario, ephrin-A1-EphA2 interaction triggered spatial reorganization of the receptor on the cell membrane into microclusters that undergo inward radial transport. In contrast, when the cells expressing EphA2 receptors contact what the authors call spatial mutations, the receptor and associated signaling molecules became equally constrained as the boundaries impede radial transport of Eph-ephrin microclusters.
Local receptor activation, however, occurred irrespective of the substrate geometry. Total internal reflection microscopy tracking of unrestricted fluorescently labeled ephrin-A1 and green fluorescent protein-labeled actin revealed an annular association of F-actin with the EphA2 clusters.
Moreover, actomyosin contractility was shown to be the driving force of radial cluster movement. Consistent with an association of F-actin with EphA2, restriction of receptor movement changed the cytoskeleton to a spread morphology with filamentous actin predominantly concentrated in lamellopodia at the cell periphery.
To establish whether the propensity to radially transport the EphA2 receptor can be used to characterize breast cancer cell lines, Salaita and colleagues determined a radial distribution function for 26 mammary cancer cell lines with different molecular and phenotypic signatures in neoplasia. The spatial organization phenotypes were then correlated with genomic and proteomic data available from these lines.
There was no correlation to the mRNA and protein expression levels of EphA2; however, an association between radial EphA2 transport and signaling pathways that are associated with invasiveness - such as ErbB, p53, integrin and mitogenactivated protein kinase - became apparent.
Selected Novel Anticancer Treatments Targeting Cell Signaling Proteins
Examples of molecularly targeted signal transduction agents include inhibitors of ras farnesylation and receptor kinase inhibitors. Approaches that target the tumor cell environment include metalloprotease and integrin inhibitors as well as antiangiogenic strategies. Such agents present a major challenge to the process of preclinical assessment. Preclinical studies must demonstrate that a candidate agent selected for clinical development acts by the desired molecular mechanism e.
The agent must also exhibit in vivo antitumor activity and a suitable therapeutic index i. Traditional end points, such as simple tumor cell killing or growth delay, are not by themselves sufficient for this contemporary need. The opportunity presented is to replace such blunt instruments with more specific end points that are tailor-made for any particular molecular target. Mechanism-based approaches require corresponding mechanism-based end points. In addition, there is pressure both in terms of unmet medical need and commercial concerns to deliver candidates for clinical development faster than ever before 4.
Companies that have already adopted aggressive time scales of years from a potential new target to a development candidate are now seeking to shorten these still further. Faster development can be achieved only by exploiting the latest technologic advances, many of which are in their infancy and rapidly changing 5. Bottlenecks exist.
In particular, bottlenecks occur with respect to understanding the biologic function of genes discovered through genomic approaches, the validation of potential drug targets, conversion of lead drugs active in vitro into agents with drug-like properties and pharmacologic activity in the intact animal, and the establishment of appropriate assays to measure specific molecular mechanisms and biologic effects.
Therefore, these are the areas that require the greatest attention in terms of technology development at the present time. Whether a drug discovery program is based on screening, a structure-based approach, or some combination of these approaches, a robust, efficient, and informative test cascade is essential This should be based on a target profile that defines the essential and desirable properties of the molecule sought e. A typical contemporary test cascade consists of a series of hierarchical assays. These assays usually begin with an initial biochemical assay on the recombinant target protein and proceed to in vitro cell tests and in vivo evaluation of pharmacokinetic and pharmacodynamic behavior to a final cancer disease model such as a human tumor xenograft Fig.
When interesting agents are discovered because of a unique cellular fingerprint or because of an association with molecular target expression, as in the National Cancer Institute's tumor panel 14 , specific assays must also be applied to confirm the mode of action and optimize the lead drug. Examples of the mode-of-action assays include analysis of the changes in phosphorylation of a particular target protein, the disruption of a protein-protein interaction, or the disruption of an appropriately engineered reporter gene system.
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A frequent point of failure in drug discovery programs is poor pharmacokinetic behavior when the agent progresses from testing in cell culture to testing in the intact animal This issue is being addressed by determining approximate pharmacokinetic properties of drug leads by the cocktail or cassette dosing approach that involves administering compounds at low doses in mixtures, followed by high-sensitivity high-performance liquid chromatography-mass to charge ratio-mass spectroscopy analysis Surrogate markers of mode of action or biologic effect can be used alongside or instead of the hollow-fiber test.
For example, an anti-inflammatory assay could be used to measure effective blockade of the phosphatidylinositol 3-kinase pathway. The use of such pharmacokinetic and surrogate pharmacodynamic end points provides the advantages of rapid and potentially cost-effective turnaround of in vivo results and a less demanding hurdle during the in vivo lead optimization phase compared with a full-blown disease model.
However, suitably optimized compounds will normally require testing in a more challenging biologic system. Human tumor xenografts are often used for this purpose 18 , but these models can be criticized as poorly predictive of the human disease although it is, in fact, too early to judge this for the newer molecular targets These concerns may be especially relevant where the drug effect sought inhibits angiogenesis or metastasis.
A case can be made for the construction and use of more complex disease models, such as orthotopic systems 20 or transgenic animals 21 , as was done for evaluating ras farnesylation inhibitors In some cases, well-characterized syngeneic models may have advantages over human tumor xenografts. It should be remembered that regressions might not be seen with signal transduction or cell cycle inhibitors, unless of course the agent induces apoptosis. In all situations, a decision to proceed to the clinic will require some evidence of activity in an in vivo disease model such as a human tumor xenograft.
Toxicologic evaluation is essential to ensure acceptable safety, to identify organs at risk, and to determine a safe phase I starting dose. However, because excessive preclinical evaluation can cause delay and is often poorly predictive, organizations such as the Cancer Research Organization and the European Organization for Research and Treatment of Cancer favor entering clinical trials after a relatively simple program of rodent-only toxicology testing 23 , There are major advantages to be gained if the mode of action and surrogate assays, ideally measuring biochemical effects as close as possible to the precise molecular locus, are capable of translation for use in the early clinical studies because they smooth the transition from preclinical assessment to human investigation.
In this regard, noninvasive assays, such as magnetic resonance spectroscopy, magnetic resonance imaging MRI , and positron emission tomography PET , are especially attractive Phase I trial design should incorporate the evaluation of safety and toxicity with a measure of the outcome at the molecular level. The ideal situation for proceeding from the laboratory to a clinical trial after a validated target effect is something that must be coordinated between the laboratory scientists and the clinical researchers before the initiation of the clinical program Table 1.
Accelerated phase I designs 26 are becoming widely used for drugs that have conventional cytotoxic activity, and there is a wealth of clinical experience to justify this approach.
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However, most novel agents e. Drugs acting on highly specific targets that may be differentially expressed or activated in cancer cells may result in low normal tissue toxicity; therefore, increasing a dose to normal tissue tolerance may be an irrelevant end point. It may also be reasonable to establish the concentration of drug required to inhibit the target and then to use that dose in target-directed trials i.
Phase I design may require fixed dosing or possibly the random assignment of patients to different dose levels with doses chosen from the preclinical studies and based on the minimum dose required to inhibit the target. The key end point in phase I trials for targeted therapies should evolve from the current idea of a MTD in normal tissue to a more suitable end point of the dose required to maximally inhibit the relevant target.
If this cannot be assessed, then all subsequent studies will be carried out blindly. With conventional drugs, clinical researchers assume that a correct dose is being used when responses and dose-limiting toxicity in normal tissues are seen. With molecularly targeted therapies, these end points may not be reached unless there are markers of target activity that are direct or indirect.
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Clearly, this is not necessary if responses occur in phase I, as with the bryostatin, the partial agonist of protein kinase C 27 , and this recommendation is most relevant for drugs that do not produce a response e. In some of these trials, the rate of change of the relevant markers has been used as the end point 1. The goal may not be target inhibition but rather subtle target modulation.
Several strategies have been developed to approach the problem of measuring target modulation. These begin to overlap with the current definition of a phase II trial. One approach would be the preoperative administration of drug and subsequent analysis of the drug concentration and target activity in the tumor. Many tumor types are managed with preoperative biopsies control sample ; for these cases, a comparison with non-drug-treated controls is possible. Similarly, tumors that produce effusions or metastases that allow easy retrieval of cells could be used.
The prerequisite for a tissue biopsy and a demonstration of target activity would lengthen the time to complete trials and would increase the costs. Nevertheless, all subsequent trials and drug development would benefit from the knowledge that the recommended dose was correct and inhibited the target for which it was designed. Our standard histologic groupings may need to be replaced with more sophisticated stratification of tumors based on molecular genetics tests.
Phase II trials of targeted therapy may not use histologic eligibility criteria but rather may enroll patients with tumors from different sites that express similar molecular markers e. There are many challenges to successfully incorporating tumor tissue analysis into the design of a clinical trial.
Overcoming these challenges requires the commitment of oncologists, pathologists, human subject protection committees, and patients; substantial additional funding; and standardization of tissue sampling to ensure that the tumor is being measured.
Two other less invasive approaches can be used. The first approach uses peripheral leukocytes, which possess many receptors and signaling pathways relevant to novel agents. PET scanning can measure blood flow, thymidine metabolism, and glucose uptake PET labeling drugs can show tissue concentrations of the drug achieved at tumor sites. An MRI can also show vascular permeability 34 and blood flow. Doppler techniques, particularly applied to breast cancer tumors, provide another useful way to assess blood flow.
Many newer agents directly or indirectly modulate tumor angiogenesis 35 , therefore, inhibition of angiogenesis is a valid biologic end point. These techniques, however, need to be rigorously assessed for reproducibility and validated, which should occur in parallel with the clinical studies e. Use of these techniques toward the end of phase I should continue into phase II to allow assessment of long-term treatment. Issues of scheduling, including long infusions, daily treatments, or long-term intermittent dosing, must be considered, and these issues may require novel pharmacokinetic modeling and sampling.
Moreover, issues such as tissue accumulation of drug may be more relevant than plasma peaks and may not be easily measured.
Cell Signaling and Intracellular Trafficking in Cancer Biology: Interplay, Targeting and Therapy -
Preclinical models suggest that many of the novel agents may stabilize tumor growth rather than produce tumor regression. This outcome may be valuable to patients and may be worthwhile when applied to adjuvant therapy aimed at dividing cells. Long-term dosing may be necessary to observe and maintain stabilization or response. Because many patients in phase I trials receive therapy for only a brief time, often only 1 or 2 months, the information required for evidence of biologic activity and phase II dosing may not be available from the initial dose studies.
Overlap and continuous progression to phase II should be in the trial design so that fitter patients can be treated for a longer period. It is not known whether molecularly targeted therapies will be active alone, combined with cytotoxic agents, or combined with other targeted therapies. Each of these permutations cannot be fully explored with the current preclinical and phase I design without delaying the answers well into the next century. Communication between laboratory researchers and clinical investigators is necessary to design batteries of tests that will allow early clinical trials of these therapies, based on preclinical data.
Certainly, results of preclinical studies have predicted some of our current clinical combinations such as 5-fluorouracil-folinic acid and gemcitabine-cisplatin and may be a model for synergistic interactions that may not be predicted a priori. Novel formulations that contain two or more agents may be desirable and may allow the initiation of early trials without the difficulties inherent in coordinating the simultaneous use of a number of experimental therapies owned by different companies.
After phase I trials are completed, the main goal of subsequent clinical research is to define as accurately and efficiently as possible the efficacy of the new agent in a variety of malignancies. Efficacy in this context is defined as increased survival, cure rates, or quality of life. Traditionally, phase II trials have been used to screen new agents for evidence of biologic activity in multiple types of tumors before proceeding into large phase III trials in which efficacy may be convincingly demonstrated.
Although not all of the agents that are selected on the basis of response results in phase II trials have been subsequently shown to improve survival, sufficient numbers have done so to validate response as a reliable phase II end point. If stasis of tumor growth rather than tumor regression is the anticipated clinical outcome, screening agents for efficacy with standard phase II response end points may be unrewarding.
Two general approaches are possible: 1 to identify surrogate end points for efficacy in phase II besides tumor response or 2 to move the new agent directly from phase I into randomized studies. There has been much interest in the first of these suggestions: the identification of alternative end points to response that may be used to appropriately select or reject a new drug in a phase II design. However, it is important to note that any proposed alternative end point must be viewed as being experimental because, until now, only response has been used to identify agents that have an impact on survival.
Possible candidate end points are as follows: time to progression, changes in tumor markers, measures of target inhibition, PET scanning, proportion of patients with early disease progression, and assessment of clinical benefit. Although median time to disease progression has the advantage of being a well-described and standardized end point in randomized trials, it is difficult to interpret when there is no control group and when patient numbers are limited, as is the case in a phase II setting.
Tumor markers also share the advantage of being well described and easily measured. The problem with using markers as an end point to select active agents is that the use of markers alone to identify active agents has never been validated. Rustin et al. For cytostatic agents in general, it is not known whether a tumor marker will decrease with treatment and, if it does not, whether the agent can still cause tumor stasis. Another report 37 has suggested that a change in the rate of increase in a relevant marker may be a useful end point for identifying an active agent. In studies of a metalloproteinase inhibitor, patients were observed to show a decline in the pretreatment rate of increase of the serum tumor marker, an effect that also seemed to demonstrate a dose-response relationship.
Randomized trials based on these data are proceeding for patients with ovarian and pancreatic cancers, and the results of these studies may contribute to validation of this end point. Furthermore, before a tumor marker is accepted as a surrogate for drug activity, it must be demonstrated that the drug under study cannot alter levels of the marker through changes unrelated to tumor burden. Such a change would be the secretion of the marker from the cell, which has been observed for some agents Measure of target inhibition is appealing from a scientific point of view.
However, because many novel agents target molecules that are only hypothetically linked to tumor growth, the inhibition of these target molecules has not yet been shown to be clinically effective. Measures of target inhibition preferably in tumor tissue are a more relevant end point for phase I trials where optimal dosing is the goal. PET scanning is a nuclear medicine technique that uses radiopharmaceuticals such as 2-[ 18 F]fluorodeoxy- d -glucose to detect differences in tissue function and metabolism. PET scanning can predict responses early when there is corresponding evidence of clinical regression Will it, however, assess changes in tumors that result from effective cytostatic therapy when a response is not documented?
This is possible, but remains to be shown. If PET scanning can only anticipate tumor regression, then this costly test is not really an alternative to response. Progression rate i. This analysis helps to define signaling rewiring and adaptive responses. Ongoing studies:. The non-receptor tyrosine kinase PYK2 and its downstream signaling pathways that regulate cell proliferation, migration and invasion. PYK2 contains several structural domains including, an N-terminal FERM domain that interacts with the Nir proteins Nir1, Nir2 and Nir3 , a central kinase domain, two short proline rich domains brown that interact with multiple SH3 containing proteins, and a C-terminal focal adhesion targeting FAT domain.