Patrick C. Ma

c-Met: Structure, functions and potential for therapeutic inhibition

Studies on signal transduction pathways have generated various promising molecular targets for therapeutic inhibition in cancer therapy. Receptor tyrosine kinases represent an important class of such therapeutic targets. c-Met is a receptor tyrosine kinase that has been shown to be overexpressed and/or mutated in a variety of malignancies. A number of c-Met activating mutations, many of which are located in the tyrosine kinase domain, have been detected in various solid tumors and have been implicated in invasion and metastasis of tumor cells. It is known that stimulation of c-Met via its natural ligand, hepatocyte growth factor (also known as scatter factor, HGF/SF) results in a plethora of biological and biochemical effects in the cell. Activation of c-Met signaling can lead to scattering, angiogenesis, proliferation, enhanced cell motility, invasion, and eventual metastasis. In this review, the role of c-Met dysregulation in tumor progression and metastasis is discussed in detail with particular emphasis on c-Met mutations. Moreover, we summarize current knowledge on various pathways of c-Met signal transduction, highlighting the central role in the cytoskeletal functions. In this summary is included recent data in our laboratory indicating that phosphorylation of focal adhesion proteins, such as paxillin, p125FAK, and PYK2, occurs in response to c-Met stimulation in lung cancer cells. Most importantly, current data on c-Met suggest that when mutated or overexpressed in malignant cells, c-Met would serve as an important therapeutic target.

Functional Expression and Mutations of c-Met and Its Therapeutic Inhibition with SU11274 and Small Interfering RNA in Non-Small Cell Lung Cancer

Non-small cell lung cancer (NSCLC) is a difficult disease to treat. The c-Met receptor is an attractive potential target for novel therapeutic inhibition in human cancers. We provide strong evidence that c-Met is overexpressed, activated, and sometimes mutated in NSCLC cell lines and tumor tissues. Expression of c-Met was found in all (100%) of the NSCLC tumor tissues examined (n = 23) and most (89%) of the cell lines (n = 9). Sixty-one percent of tumor tissues strongly expressed total c-Met, especially adenocarcinoma (67%). Specific expression of phospho-Met (p-Met) [Y1003] and [Y1230/1234/1235] was seen by immunohistochemistry. p-Met expression was preferentially observed at the NSCLC tumor invasive fronts. c-Met alterations were identified within the semaphorin domain (E168D, L299F, S323G, and N375S) and the juxtamembrane domain (R988C, R988C + T1010I, S1058P, and alternative splice product skipping entire juxtamembrane domain) of a NSCLC cell line and adenocarcinoma tissues. We validated c-Met as potential therapeutic target using small interfering RNA down-regulation of the receptor expression by 50% to 60% in NSCLC cells. This led to inhibition of p-Met and phospho-AKT and up to 57.1 +/- 7.2% cell viability inhibition at 72 hours. The selective small molecule inhibitor of c-Met SU11274 inhibited cell viability in c-Met-expressing NSCLC cells. SU11274 also abrogated hepatocyte growth factor-induced phosphorylation of c-Met and its downstream signaling. Here, we provide first direct evidence by small interfering RNA targeting and small molecule inhibitor that c-Met is important in NSCLC biology and biochemistry. These results indicate that c-Met inhibition will be an important therapeutic strategy against NSCLC to improve its clinical outcome.

Role of the hepatocyte growth factor receptor, c-Met, in oncogenesis and potential for therapeutic inhibition

Receptor tyrosine kinases have become important therapeutic targets for anti-neoplastic molecularly targeted therapies. c-Met is a receptor tyrosine kinase shown to be over-expressed and mutated in a variety of malignancies. Stimulation of c-Met via its ligand hepatocyte growth factor also known as scatter factor (HGF/SF), leads to a plethora of biological and biochemical effects in the cell. There has been considerable knowledge gained on the role of c-Met-HGF/SF axis in normal and malignant cells. This review summarizes the structure of c-Met and HGF/SF and their family members. Since there are known mutations of c-Met in solid tumors, particularly in papillary renal cell carcinoma, we have summarized the various mutations and over-expression of c-Met known thus far. Stimulation of c-Met can lead to scattering, angiogenesis, proliferation, enhanced cell motility, invasion, and eventual metastasis. The biological functions altered by c-Met are quite unique and described in detail. Along with biological functions, various signal transduction pathways, including the cytoskeleton are altered with the activation of c-Met-HGF/SF loop. We have recently shown the phosphorylation of focal adhesion proteins, such as paxillin and p125FAK in response to c-Met stimulation in lung cancer cells, and this is detailed here. Finally, c-Met when mutated or over-expressed in malignant cells serves as an important therapeutic target and the most recent data in terms of inhibition of c-Met and downstream signal transduction pathways is summarized.

Functional analysis of c-Met/hepatocyte growth factor pathway in malignant pleural mesothelioma

c-Met receptor tyrosine kinase (RTK) has not been extensively studied in malignant pleural mesothelioma (MPM). In this study, c-Met was overexpressed and activated in most of the mesothelioma cell lines tested. Expression in MPM tissues by immunohistochemistry was increased (82%) in MPM in general compared with normal. c-Met was internalized with its ligand hepatocyte growth factor (HGF) in H28 MPM cells, with robust expression of c-Met. Serum circulating HGF was twice as high in mesothelioma patients as in healthy controls. There was a differential growth response and activation of AKT and extracellular signal-regulated kinase 1/2 in response to HGF for the various cell lines. Dose-dependent inhibition (IC50 < 2.5 micromol/L) of cell growth in mesothelioma cell lines, but not in H2052, H2452, and nonmalignant MeT-5A (IC50 > 10 micromol/L), was observed with the small-molecule c-Met inhibitor SU11274. Furthermore, migration of H28 cells was blocked with both SU11274 and c-Met small interfering RNA. Abrogation of HGF-induced c-Met and downstream signaling was seen in mesothelioma cells. Of the 43 MPM tissues and 7 cell lines, we have identified mutations within the semaphorin domain (N375S, M431V, and N454I), the juxtamembrane domain (T1010I and G1085X), and an alternative spliced product with deletion of the exon 10 of c-Met in some of the samples. Interestingly, we observed that the cell lines H513 and H2596 harboring the T1010I mutation exhibited the most dramatic reduction of cell growth with SU11274 when compared with wild-type H28 and nonmalignant MeT-5A cells. Ultimately, c-Met would be an important target for therapy against MPM.

A Phase II Study of ABT-751 in Patients with Advanced Non-small Cell Lung Cancer

Purpose: To determine the tolerability and efficacy of ABT-751, an oral antimitotic agent that inhibits polymerization of microtubules, in patients with advanced taxane-refractory non-small cell lung carcinoma (NSCLC). Patients and Methods: Eligibility was limited to patients with recurrent or metastatic NSCLC who had received one to two cytotoxic chemotherapy regimens, had a performance status of zero to one, and adequate organ function. Treatment included ABT-751 200 mg daily for 21 consecutive days, followed by 7 days off drug. Objectives were to determine response rate, time to tumor progression, survival, and tolerability of ABT-751. Results: All 35 enrolled patients were assessable for survival, response, and tolerability. Median time to tumor progression and overall survival were 2.1 and 8.4 months, respectively. The objective response rate was 2.9%. One patient achieved a partial response that was ongoing 567 days after initial documentation. Treatment was well tolerated; fatigue, constipation, and dehydration were the only treatment related, grade three adverse events occurring in more than one patient. Incidence of grade 3/4 hematologic and blood chemistry toxicities was acceptable, and ABT-751 was not associated with myelosuppression. Conclusions: ABT-751 associated toxicity was acceptable. The median time to progression and overall survival as demonstrated for ABT-751 were comparable to other agents considered active in this patient population and to current treatments approved for second-line NSCLC. The novel antimitotic targeting of ABT-751 in combination with the compound’s acceptable nonmyelosuppressive toxicity profile and efficacy similar to agents currently in use in this setting, warrant further evaluation of this compound in combination with other cytotoxic agents in advanced NSCLC.

Fibronectin enhances viability and alters cytoskeletal functions (with effects on the phosphatidylinositol 3‐kinase pathway) in small cell lung cancer

Small cell lung cancer (SCLC) is a rapidly progressive disease with ultimate poor outcome. SCLC has been shown to interact closely with the stromal and extracellular matrix (ECM) components of the diseased host. ECM consists of type I/IV collagen, laminin, vitronectin, and fibronectin (FN) among others. Herein, we investigated the behavior of a SCLC cell line (NCI‐H446) on FN‐coated surface. Over a course of 72 h, FN (10 ?g/ml) caused both increased survival and proliferation of NCI‐H446 cells. Survival under serum‐starved conditions increased 1.44‐fold and proliferation in the presence of fetal calf serum increased by 1.30‐fold. The phosphatidylinositol 3‐kinase (PI3‐K) inhibitor LY294002 reduced both survival and proliferation of NCI‐H446 cells (0.48‐ and 0.27‐fold, respectively), even on FN‐coated surface. We next determined the effects of FN on cytoskeletal function such as cell motility/morphology and adhesion. Over a course of 24 h, FN reduced aggregation of NCI‐H446 cells and induced flattened cellular morphology with neurite‐like projections after 1 h, however, in the presence of LY294002, the cells rounded up. Adhesion of NCI‐H446 cells also increased with FN (4.47‐ fold) which was abrogated with LY294002 treatment. This correlated with phosphorylation of the cytoskeletal protein p125FAK, on Tyr397, Tyr861 and Ser843 residues with FN. Even in the presence of LY294002, these serine/tyrosine residues were still phosphorylated on FN‐coated surface. In contrast, the focal adhesion protein paxillin was not phosphorylated at Tyr31 with FN. In summary, FN stimulation of SCLC cells leads to enhancement of viability and change in cytoskeletal function that are partially mediated through the PI3‐K pathway.

Therapeutic Targeting of the Receptor Tyrosine Kinase Met

There are different options to inhibit Met activation and these are in part limited depending on the transforming phenotype. For example, inhibiting ligand/receptor interactions may be a useful approach when inhibiting a paracrine/autocrine mechanism but will show little effect on a gain-of-function mutation of Met. It still remains unknown as to how much Met inhibition is required to achieve clinically beneficial anti-tumor and anti-metastatic results. The answer may potentially be different depending upon whether the ‘tumorigenic culprit’ lies with Met expression or mutations of the RTK. It will be necessary to further study and optimize targeted approaches for clinical studies. Another intriguing area of research, with specific Met inhibitors available, would be to test the drug efficaciesagainst different mutant forms of the Met oncoprotein. It is possible that some mutations of Met may cause resistance, or conversely a greater susceptibility, to the inhibitors. Among various strategies to inhibit HGF/Met signaling, developing specific small molecule inhibitors against Met may have the greatest potential in molecularly targeted anti-cancer therapy. However, lessons learned from Gleevec (imatinib mesylate) treatment in chronic myelogenous leukemia demonstrate that drug resistance is a real issu therapeutically. It will therefore be necessary to furthercharacterize the signaling mechanisms behind Met-mediated proliferation and other biological effects to identify additional molecules for targeted therapies.