Met Ligand Research Articles

Coagulation and angiogenic gene expression profiles are defined by molecular subgroups of medulloblastoma: evidence for growth factor‐thrombin cross‐talk

The coagulation system becomes activated during progression and therapy of high-grade brain tumors. Triggering tissue factor (F3/TF) and thrombin receptors (F2R/PAR-1) may influence the vascular tumor microenvironment and angiogenesis irrespective of clinically apparent thrombosis. These processes are poorly understood in medulloblastoma (MB), in which diverse oncogenic pathways define at least four molecular disease subtypes (WNT, SHH, Group 3 and Group 4). We asked whether there is a link between molecular subtype and the network of vascular regulators expressed in MB. Using R2 microarray analysis and visualization platform, we mined MB datasets for differential expression of vascular (coagulation and angiogenesis)-related genes, and explored their link to known oncogenic drivers. We evaluated the functional significance of this link in DAOY cells in vitro following growth factor and thrombin stimulation. The coagulome and angiome differ across MB subtypes. F3/TF and F2R/PAR-1 mRNA expression are upregulated in SHH tumors and correlate with higher levels of hepatocyte growth factor receptor (MET). Cultured DAOY (MB) cells exhibit an up-regulation of F3/TF and F2R/PAR-1 following combined SHH and MET ligand (HGF) treatment. These factors cooperate with thrombin, impacting the profile of vascular regulators, including interleukin 1β (IL1B) and chondromodulin 1 (LECT1). Coagulation pathway sensors (F3/TF, F2R/PAR-1) are expressed in MB in a subtype-specific manner, and may be functionally linked to SHH and MET circuitry. Thus coagulation system perturbations may elicit subtype/context-specific changes in vascular and cellular responses in MB.

Abstract 4513: IMGN289, an EGFR-targeting antibody-drug conjugate, is effective against tumor cells that are resistant to EGFR tyrosine kinase inhibitors

Abstract EGFR is one of the oncogenic drivers in non-small-cell lung adenocarcinoma (NSCLC AC). EGFR tyrosine kinase inhibitors (TKIs), such as erlotinib, gefitinib and afatinib, show remarkable single agent activity in NSCLC AC with EGFR mutations. However, a majority of the tumors develop resistance within twelve months. T790M secondary EGFR mutation and activation of alternative signaling pathways, such as MET, are the two most common resistance mechanisms. To address this clinical challenge, we developed IMGN289, an EGFR antibody-drug conjugate consisting of the novel J2898A antibody (Ab), which selectively binds to and inhibits EGFR-driven tumor cell growth, conjugated to the maytansinoid DM1, a potent anti-microtubule agent, via the SMCC thioether linker. IMGN289 showed significantly enhanced cytotoxic activity, relative to cetuximab or unconjugated J2898A, against tumor cell line models that are dependent on EGFR signaling (Chittenden et al., AACR 2013). To evaluate the ability of IMGN289 to overcome mechanisms of EGFR TKI resistance, IMGN289 activity was tested in erlotinib-resistant EGFR-mutant cell lines which overexpressed MET (HCC827-ER) or carry the T790M EGFR mutation (HCC827-EPR) (Suda et al., Clin Cancer Res 2010). Despite variable levels of EGFR expression in these cell lines, (193,000, 32,500 and 422,000 antigens per cell in HCC827, HCC827-ER and HCC827-EPR, respectively), IMGN289 showed comparable cytotoxic activities against these cell lines in vitro. In addition to MET overexpression, the MET pathway can also be activated by HGF, the MET ligand. While addition of HGF made the HCC827 cell line resistant to EGFR TKIs (Yano et al., Cancer Res 2008) and decreased growth inhibition by cetuximab, IMGN289 activity was unaffected. Additionally, we generated an erlotinib-resistant HCC827-ER-E4 cell line by exposing the EGFR-mutant HCC827 cells to increasing concentrations of erlotinib for ∼6 months, followed by subcloning. The HCC827-ER-E4 cell line expressed mesenchymal markers and acquired a mesenchymal phenotype. It expressed lower levels of EGFR, HER3 and MET compared to the parental cell line and showed no loss of PTEN. In the presence of erlotinib, HCC827-ER-E4 cells retained high levels of pAKT and pERK, suggesting that activation of alternative signaling pathways may be responsible for the erlotinib resistance. In vitro cytotoxic assays showed that IMGN289 was equally cytotoxic to the parental and the HCC827-ER-E4 cell line. In conclusion, IMGN289 demonstrated EGFR-targeted cytotoxicity against NSCLC cell lines that harbor some of the most common mechanisms of resistance to EGFR TKIs. IMGN289 represents a promising novel candidate for treatment of EGFR-expressing NSCLC AC that has acquired resistance to EGFR TKIs. Citation Format: Yulius Y. Setiady, Ling Dong, Anna Skaletskaya, Jan Pinkas, Robert J. Lutz, John M. Lambert, Thomas Chittenden. IMGN289, an EGFR-targeting antibody-drug conjugate, is effective against tumor cells that are resistant to EGFR tyrosine kinase inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4513. doi:10.1158/1538-7445.AM2014-4513

The potential roles of hepatocyte growth factor (HGF)-MET pathway inhibitors in cancer treatment.

MET is located on chromosome 7q31 and is a proto-oncogene that encodes for hepatocyte growth factor (HGF) receptor, a member of the receptor tyrosine kinase (RTK) family. HGF, also known as scatter factor (SF), is the only known ligand for MET. MET is a master regulator of cell growth and division (mitogenesis), mobility (motogenesis), and differentiation (morphogenesis); it plays an important role in normal development and tissue regeneration. The HGF-MET axis is frequently dysregulated in cancer by MET gene amplification, translocation, and mutation, or by MET or HGF protein overexpression. MET dysregulation is associated with an increased propensity for metastatic disease and poor overall prognosis across multiple tumor types. Targeting the dysregulated HGF-MET pathway is an area of active research; a number of monoclonal antibodies to HGF and MET, as well as small molecule inhibitors of MET, are under development. This review summarizes the key biological features of the HGF-MET axis, its dysregulation in cancer, and the therapeutic agents targeting the HGF-MET axis, which are in development.

Increased Epidermal Growth Factor Receptor (EGFR) Associated with Hepatocyte Growth Factor (HGF) and Symptom Severity in Children with Autism Spectrum Disorders (ASDs).

BACKGROUNDOne in 88 children in the US is thought to have one of the autism spectrum disorders (ASDs). ASDs are characterized by social impairments and communication problems. Growth factors and their receptors may play a role in the etiology of ASDs. Research has shown that epidermal growth factor receptor (EGFR) activation is associated with nerve cell development and repair. This study was designed to measure plasma levels of EGFR in autistic children and correlate these levels with its ligand, epidermal growth factor, other related putative biomarkers such as hepatocyte growth factor (HGF), the ligand for MET (MNNG HOS transforming gene) receptor, as well as the symptom severity of 19 different behavioral symptoms.SUBJECTS AND METHODSPlasma EGFR concentration was measured in 33 autistic children and 34 age- and gender-similar neurotypical controls, using an enzyme-linked immunosorbent assay. Plasma EGFR levels were compared to putative biomarkers known to be associated with EGFR and MET and severity levels of 19 autism-related symptoms.RESULTSWe found plasma EGFR levels significantly higher in autistic children, when compared to neurotypical controls. EGFR levels correlated with HGF and high-mobility group protein B1 (HMGB1) levels, but not other tested putative biomarkers, and EGFR levels correlated significantly with severity of expressive language, conversational language, focus/attention, hyperactivity, eye contact, and sound sensitivity deficiencies.CONCLUSIONSThese results suggest a relationship between increased plasma EGFR levels and designated symptom severity in autistic children. A strong correlation between plasma EGFR and HGF and HMGB1 suggests that increased EGFR levels may be associated with the HGF/Met signaling pathway, as well as inflammation.

Fibroblast-secreted hepatocyte growth factor mediates epidermal growth factor receptor tyrosine kinase inhibitor resistance in triple-negative breast cancers through paracrine activation of Met

IntroductionEpidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown clinical efficacy in lung, colon, and pancreatic cancers. In lung cancer, resistance to EGFR TKIs correlates with amplification of the hepatocyte growth factor (HGF) receptor tyrosine kinase Met. Breast cancers do not respond to EGFR TKIs, even though EGFR is overexpressed. This intrinsic resistance to EGFR TKIs in breast cancer does not correlate with Met amplification. In several tissue monoculture models of human breast cancer, Met, although expressed, is not phosphorylated, suggesting a requirement for a paracrine-produced ligand. In fact, HGF, the ligand for Met, is not expressed in epithelial cells but is secreted by fibroblasts in the tumor stroma. We have identified a number of breast cancer cell lines that are sensitive to EGFR TKIs. This sensitivity is in conflict with the observed clinical resistance to EGFR TKIs in breast cancers. Here we demonstrate that fibroblast secretion of HGF activates Met and leads to EGFR/Met crosstalk and resistance to EGFR TKIs in triple-negative breast cancer (TNBC).MethodsThe SUM102 and SUM149 TNBC cell lines were used in this study. Recombinant HGF as well as conditioned media from fibroblasts expressing HGF were used as sources for Met activation. Furthermore, we co-cultured HGF-secreting fibroblasts with Met-expressing cancer cells to mimic the paracrine HGF/Met pathway, which is active in the tumor microenvironment. Cell growth, survival, and transformation were measured by cell counting, clonogenic and MTS assays, and soft agar colony formation, respectively. Student's t test was used for all statistical analysis.ResultsHere we demonstrate that treatment of breast cancer cells sensitive to EGFR TKIs with recombinant HGF confers a resistance to EGFR TKIs. Interestingly, knocking down EGFR abrogated HGF-mediated cell survival, suggesting a crosstalk between EGFR and Met. HGF is secreted as a single-chain pro-form, which has to be proteolytically cleaved in order to activate Met. To determine whether the proteases required to activate pro-HGF were present in the breast cancer cells, we utilized a fibroblast cell line expressing pro-HGF (RMF-HGF). Addition of pro-HGF-secreting conditioned fibroblast media to TNBC cells as well as co-culturing of TNBC cells with RMF-HGF fibroblasts resulted in robust phosphorylation of Met and stimulated proliferation in the presence of an EGFR TKI.ConclusionsTaken together, these data suggest a role for Met in clinical resistance to EGFR TKIs in breast cancer through EGFR/Met crosstalk mediated by tumor-stromal interactions.

Mutant p53 enhances MET trafficking and signalling to drive cell scattering and invasion.

Tumour-derived mutant p53 proteins promote invasion, in part, by enhancing Rab coupling protein (RCP)-dependent receptor recycling. Here we identified MET as an RCP-binding protein and showed that mutant p53 promoted MET recycling. Mutant p53-expressing cells were more sensitive to hepatocyte growth factor, the ligand for MET, leading to enhanced MET signalling, invasion and cell scattering that was dependent on both MET and RCP. In cells expressing the p53 family member TAp63, inhibition of TAp63 also lead to cell scattering and MET-dependent invasion. However, in cells that express very low levels of TAp63, the ability of mutant p53 to promote MET-dependent cell scattering was independent of TAp63. Taken together, our data show that mutant p53 can enhance MET signalling to promote cell scattering and invasion through both TAp63-dependent and -independent mechanisms. MET has a predominant role in metastatic progression and the identification of mechanisms through which mutations in p53 can drive MET signalling may help to identify and direct therapy.

Met receptor tyrosine kinase signals through a cortactin-Gab1 scaffold complex, to mediate invadopodia

SummaryInvasive carcinoma cells form actin-rich matrix-degrading protrusions called invadopodia. These structures resemble podosomes produced by some normal cells and play a crucial role in extracellular matrix remodeling. In cancer, formation of invadopodia is strongly associated with invasive potential. Although deregulated signals from the receptor tyrosine kinase Met (also known as hepatocyte growth factor are linked to cancer metastasis and poor prognosis, its role in invadopodia formation is not known. Here we show that stimulation of breast cancer cells with the ligand for Met, hepatocyte growth factor, promotes invadopodia formation, and in aggressive gastric tumor cells where Met is amplified, invadopodia formation is dependent on Met activity. Using both GRB2-associated-binding protein 1 (Gab1)-null fibroblasts and specific knockdown of Gab1 in tumor cells we show that Met-mediated invadopodia formation and cell invasion requires the scaffold protein Gab1. By a structure–function approach, we demonstrate that two proline-rich motifs (P4/5) within Gab1 are essential for invadopodia formation. We identify the actin regulatory protein, cortactin, as a direct interaction partner for Gab1 and show that a Gab1–cortactin interaction is dependent on the SH3 domain of cortactin and the integrity of the P4/5 region of Gab1. Both cortactin and Gab1 localize to invadopodia rosettes in Met-transformed cells and the specific uncoupling of cortactin from Gab1 abrogates invadopodia biogenesis and cell invasion downstream from the Met receptor tyrosine kinase. Met localizes to invadopodia along with cortactin and promotes phosphorylation of cortactin. These findings provide insights into the molecular mechanisms of invadopodia formation and identify Gab1 as a scaffold protein involved in this process.

Abstract A233: Cabozantinib (XL184), a dual MET-VEGFR2 inhibitor, blocks osteoblastic and osteolytic progression of human prostate cancer xenografts in mouse bone.

Abstract Background: Prostate cancer bone metastases are associated with high levels of MET expression, and both HGF, the only known ligand for MET, and VEGF appear to direct crosstalk between tumor cells, osteoblasts (OBs), and osteoclasts (OCs). Cabozantinib (cabo), a dual MET-VEGFR2 inhibitor, has shown clinical activity in patients with metastatic castration-resistant prostate cancer (CRPC), where a complete or partial resolution of lesions on bone scans, reduced pain, and soft tissue tumor regression were observed in the majority of patients studied. Therefore, the preclinical effects of cabo were studied in the human CRPC bone xenograft model ARCaPM, which expresses both MET and the VEGF co-receptor neuropilin-1. In addition, the effects of cabo on the differentiation and activity of human OCs and mouse OBs were studied in vitro. Methods: ARCaPM cells were injected bilaterally into the tibiae of nude mice on Day 1, and on Day 31 animals received either cabo at 10 or 30 mg/kg (mpk) or vehicle once-daily for 7 weeks (wks). Animals (n=10 for each dose group) were sacrificed at the end of the treatment period and X-ray images of the tibiae were taken. Five representative tibiae per group were also scanned and analyzed by a Scanco 40 micro-CT instrument. In addition, one tibia from each mouse was fixed, decalcified and embedded for histology and histomorphometry analyzes. The OC culture system consisted of CD34+ cells derived from human bone marrow that were cultured on bovine bone slices in the presence of growth factors including M-CSF and RANK-L. The OB culture system used mouse KS483 cells that differentiate into OBs capable of forming mineralized bone nodules. Results: X-ray and micro-CT imaging of tibiae harvested after the 7 wk treatment period indicated that cabo treatment blocked both the osteoblastic and osteolytic progression of ARCaPM xenograft tumors in bone, with concomitant increases in bone mineral density. Histomorphometry data indicated that tumors were present in 70% (7/10) of the tibiae in the control group but only 30% (3/10) and 20% (2/10) in the 10 mpk and 30 mpk cabo groups, respectively. Consistent with its anti-tumor effect in the bone, cabo treatment decreased the ratio of tumor to tissue area and increased the bone area relative to the tissue area in the analyzed tibia sections compared to vehicle. Histological analyzes indicated an increase in the number of OBs and no change in the number of OCs along the trabecular bone perimeter of tibiae from cabo-treated animals relative to vehicle controls. In the in vitro studies, cabo treatment resulted in reduced OC differentiation in a dose dependent manner but did not affect the ability of mature OCs to resorb bone. In contrast, in the OB studies cabo treatment resulted in a biphasic effect inducing OB differentiation and bone forming activity at the lower doses while reducing these parameters at higher doses. Conclusions: Cabo treatment resulted in diverse effects on the differentiation and function of OBs and OCs in vitro, and blocked both osteoblastic and osteolytic progression of ARCaPM xenograft tumors in bone. These data demonstrate a substantial impact of cabo on tumor cells, OBs, and OCs consistent with the observed clinical results in patients with CRPC. Studies to characterize the molecular mechanisms underlying these effects are underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A233.

Inhibition of MET Receptor Tyrosine Kinase and Its Ligand Hepatocyte Growth Factor

Target-based therapeutic development has significantly changed cancer treatment and has generated new sense of optimism. Cancers that contain multiple genetic and epigenetic abnormalities remain addicted to one or few genes for their malignant phenotype and cell survival. Inhibition of these abnormalities via monoclonal antibodies (mAbs) and small-molecule inhibitors (SMIs) have brought about new hope in NSCLC. MET gene was initially discovered in the laboratory of George Vande Woude as part of a transforming fusion gene between transforming promoter region (TPR) and MET which was derived from a chromosomal translocation after treatment with N-methyl-N-nitro-N-nitrosoguanidine. 1 MET is located on chromosome 7, encodes for a single precursor which is post-transcriptionally digested, forming an extracellular -chain and a transmembrane s-chain linked by disulfide bonds. The ligand for MET is hepatocyte growth factor (HGF)/Scatter Factor (SF), which is synthesized as a single inactive polypeptide chain (pro-HGF/SF) and cleaved to an active, disulfide-linked dimer by serine proteases including uPA, tPA, and then binds to the MET Sema domain. 2 Ligation of the MET receptor by HGF leads to receptor dimerization and activation of its intrinsic tyrosine kinase and further autophosphorylation or phosphorylation of downstream intermediates and activation of signaling pathways. MET can be activated by mutations, autocrine/paracrine growth, overexpression by gene amplification, or decreased degradation. Germline and somatic MET gene mutations have been reported in hereditary and sporadic papillary renal cell cancers. 3‐5 Both MET gene mutations and amplifications have now been reported in other cancers, although at low frequencies but as genetic predictors of therapeutic sensitivity. 6 Expression of MET and phosphoMET has been studied in lung cancer, and recently it was shown that 40% of lung cancer tissues overexpressed MET. 7 Summary of Presentations MET as a therapeutic target in NSCLC Dr. Robert C. Doebele discussed some of the key background points surrounding MET inhibition in NSCLC. He noted that survival in NSCLC patients with 5 copies/cell is worse than those with less than 5 copies/cell and that MET gene amplification leads to EGFR tyrosine kinase resistance in EGFR mutant patients.8 Anti-HGF Abs, anti-MET Abs, and small-molecule MET TKI inhibitors are all in various stages of development, and predictive biomarkers for MET inhibitors will be important to elucidate for future trials and treatment decisions.

Methionine Ligand Lability in Bacterial Monoheme Cytochromes c: An Electrochemical Study

The direct electrochemical analysis of adsorbed redox active proteins has proven to be a powerful technique in biophysical chemistry, frequently making use of the electrode material pyrolytic "edge-plane" graphite. However, many heme-bearing proteins such as cytochromes c have been also examined systematically at alkanethiol-modified gold surfaces, and previously we reported the characterization of the redox properties of a series of bacterial cytochromes c in a side-by-side comparison of carbon and gold electrode materials. In our prior findings, we reported an unanticipated, low potential (E(m) ∼ -100 mV vs SHE) redox couple that could be analogously observed when a variety of monoheme cytochromes c are adsorbed onto carbon-based electrodes. Here we demonstrate that our prior phenomological data can be understood quantitatively in the loss of the methionine ligand of the heme iron, using the cytochrome c from Hydrogenbacter thermophilum as a model system. Through the comparison of wild-type protein with M61H and M61A mutants, in direct electrochemical analyses conducted as a function of temperature and exogenous ligand concentration, we are able to show that Met-ligated cytochromes c have a propensity to lose their Met ligand at graphite surfaces, and that energetics of this process (6.3 ± 0.2 kJ/mol) is similar to the energies associated with "foldons" of known protein folding pathways.

Model peptides provide new insights into the role of histidine residues as potential ligands in human cellular copper acquisition via Ctr1.

Cellular acquisition of copper in eukaryotes is primarily accomplished through the Ctr family of copper transport proteins. In both humans and yeast, methionine-rich "Mets" motifs in the amino-terminal extracellular domain of Ctr1 are thought to be responsible for recruitment of copper at the cell surface. Unlike yeast, mammalian Ctr1 also contains extracellular histidine-rich motifs, although a role for these regions in copper uptake has not been explored in detail. Herein, synthetic model peptides containing the first 14 residues of the extracellular domain of human Ctr1 (MDHSHHMGMSYMDS) have been prepared and evaluated for their apparent binding affinity to both Cu(I) and Cu(II). These studies reveal a high affinity Cu(II) binding site (log K = 11.0 ± 0.3 at pH 7.4) at the amino-terminus of the peptide as well as a high affinity Cu(I) site (log K = 10.2 ± 0.2 at pH 7.4) that utilizes adjacent HH residues along with an additional His or Met ligand. These model studies suggest that the histidine domains may play a direct role in copper acquisition from serum copper-binding proteins and in facilitating the reduction of Cu(II) to the active Ctr1 substrate, Cu(I). We tested this hypothesis by expressing a Ctr1 mutant lacking only extracellular histidine residues in Ctr1-knockout mouse embryonic fibroblasts. Results from live cell studies support the hypothesis that extracellular amino-terminal His residues directly participate in the copper transport function of Ctr1.

Abstract 280: Met (the HGF receptor) localizes to the nucleus in wound healing

Abstract Background: The hepatocyte growth factor (HGF)/Met signaling pathway has been shown to be important for stimulating cell proliferation, motility, invasion and metastasis. Recent work from our lab has also demonstrated patients with high levels of nuclear Met have a worse prognosis even in lymph node negative breast cancer and has also identified a 60 kDa fragment from the carboxy-terminal domain of Met that localizes to the nucleus. Methods: We developed a method for quantification of in vitro wounding of epithelial cellular monolayers that we have utilized to further characterize nuclear Met. Nuclear Met was identified by immunofluorescence with the carboxy-terminal Met specific antibody CVD13 (Invitrogen) in H1650, A431 and Caco2 cells. The Met ligand (HGF) was used to stimulate wound healing and the Pfizer Met kinase inhibitor PHA-665752 (PHA) was used to determine the dependence of activated Met for nuclear localization. Results: As previously observed, stimulating cells with HGF (50 ng/mL) increases overall wound healing and inhibition of Met kinase activity with PHA reduces overall wound healing. Using immunofluorescence we have determined nuclear Met is present at the wound edge of lung (H1650), colon (CaCo2), and skin (A431) cancer cell lines. Additionally, nuclear localization of Met is also observed in H1650 cells by immunofluorescence with a phospho-Met antibody (CST). Nuclear localization of Met is inhibited when cells are pretreated with PHA even in the presence of HGF. However, HGF stimulation alone is not sufficient to induce nuclear Met in the A549 lung cancer cell line. Finally, the 60 kDa Met fragment is absent by Western blot when H1650 cells are grown in serum-starved conditions. Conclusion: Nuclear Met is observed during the wound healing of cellular monolayers and can be inhibited with Met specific kinase inhibitors. Nuclear Met, as defined by the presence of the 60 kDa fragment, is lost in serum-starved growth conditions. Since, several pharmaceutical companies are currently developing Met-based therapies, the role of Met the nucleus may be important in future targeted cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 280.

Abstract 626: Therapeutic effect of HGF inhibitors against HGF-induced EGFR-TKI resistance in lung cancer harboring EGFR mutations

Abstract Introduction: Non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR)-activating mutations (EGFRmu) responds favorably to the EGFR tyrosine kinase inhibitors (EGFR-TKIs), gefitinib or erlotinib. However, the responders invariably acquire resistance to EGFR-TKIs. Two mechanisms, second-site point mutation that substitutes methionine for threonine at position 790 (T790M) in EGFR and amplification of MET proto-oncogene, which contribute to acquired resistance to EGFR-TKIs have been reported. As the third mechanism, we recently demonstrated that hepatocyte growth factor (HGF), specific ligand for MET, induced resistance to EGFR-TKIs. The purpose of this study was to examine whether inhibitor of HGF could overcome resistance to EGFR-TKIs induced by HGF in NSCLC cells with EGFRmu. Results: HGF induced the resistance to gefitinib and erlotinib in lung cancer cells (PC-9 and HCC827) with EGFRmu, by restoring Akt phosphorylation through MET but not EGFR or ErbB3. These NSCLC cells also became resistant to EGFR-TKIs when co-cultured in vitro with HGF-producing fibroblasts and co-injected into SCID mice. Importantly, combined use of gefitinib plus anti-HGF antibody or the HGF antagonist, NK4 successfully overcame the fibroblast-induced EGFR-TKI resistance both in vitro and in vivo. Conclusions: These findings indicate that HGF inhibitor, such as anti-HGF antibody and NK4, may be useful for controlling HGF-induced EGFR-TKI resistance in NSCLC harboring EGFRmu in humans. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 626.

Abstract A178: Role of tumor and host-derived HGF in drug resistance to EGFR inhibitors in EGFR activating mutation-positive lung cancer

Abstract Background: Non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR)-activating mutations (EGFRmu) responds favorably to the EGFR tyrosine kinase inhibitors (EGFR-TKIs), gefitinib or erlotinib. However, 25–30% of patients with EGFRmu show intrinsic resistance, and the responders invariably acquire resistance to EGFR-TKIs. Here, we examined the role of hepatocyte growth factor (HGF), a specific ligand of MET, in gefitinib resistance of NSCLC with EGFRmu. Methods: NSCLC cells, PC-9 and HCC827, with EGFRmu, but not T790M second mutation in EGFR or MET amplification, were used in this study. Sensitivity to gefitinib was determined by MTT assay. Phosphorylation of MET, EGFR, ERBB3, and the PI3K/Akt pathway were examined by Western blotting. HGF expression in 20 tumors from 16 NSCLC patients with EGFRmu who were treated with gefitinib was determined by immunohistochemistry. Results: HGF induced resistance to gefitinib by restoring Akt phosphorylation independently of ErbB3. Specific down-regulation of MET, but not ErbB3, reversed gefitinib resistance and Akt phosphorylation induced by HGF. High levels of immunoreactivity for HGF were detected in cancer cells of 3 of 3 intrinsic resistant tumors and in one of 2 tumors with acquired resistance without T790M second mutation or MET amplification, suggesting HGF-induced resistance by autocrine mechanism. The lung cancer cells also became resistant to EGFR-TKIs when co-cultured in vitro with HGF-producing fibroblasts and co-injected into SCID mice. Importantly, combined use of gefitinib plus anti-HGF antibody or the HGF antagonist, NK4 successfully overcame the fibroblast-induced EGFR-TKI resistance both in vitro and in vivo. Co-localization of fibroblasts and HGF was detected in both xenograft tumors in mouse model and lung cancer patient specimens, suggesting HGF-induced resistance by paracrine mechanism. Conclusions: The findings indicate that HGF-mediated MET activation is a novel mechanism of intrinsic and acquired gefitinib-resistance in NSCLC with EGFRmu by both autocrine and paracrine mechanisms. Therefore, inhibition of HGF-MET signaling may be considerable strategy for more successful treatment with EGFR-TKIs. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A178.

Met Over-Expression Is a Prognostic Factor for Myeloma Patients Treated with Novel Agents.

Abstract 1821Poster Board I-847 BackgroundOver-expression of the tyrosine kinase receptor Met has been reported in several solid tumors and increases cell proliferation, cell migration (scattering) and neoplastic angiogenesis. The Hepatocyte Growth Factor (HGF) is the only known Met ligand and the HGF/Met pathway seems to play a pivotal role in Multiple Myeloma (MM) pathogenesis. HGF is secreted by bone marrow stromal cells and Met is expressed at the surface of malignant plasma cells and endothelial cells. The phosphorylation of Met induced by HGF is a strong signal for cell growth and migration. The activity of the HGF/Met pathway and the heterogeneity of HGF/Met expression on MM cells suggest that the over-expression of Met could be a prognostic marker in MM patients. AimsTo investigate the role of HGF/Met pathway as prognostic marker predictive of response rate, progression-free survival (PFS) and overall survival (OS) in MM patients treated with novel agents. Methods102 newly diagnosed MM patients received the PAD-Mel100-LP-L regimen (Palumbo A, Abs 159, ASH 2008): as induction, four 21-day PAD cycles (Bortezomib, Pegylated-lyposomal-doxorubicin, Dexamethasone); as transplantation, tandem Mel100 (Melphalan 100 mg/m2) followed by stem cell support; as consolidation four 28-day LP cycles (Lenalidomide plus Prednisone) followed by Lenalidomide alone as maintenance. Samples from 47 newly diagnosed patients enrolled in this study have been analyzed. On CD138+ and CD138- cells separated from bone marrow (BM) aspirate, HGF and Met mRNA levels have been evaluated using a quantitative Real-Time PCR (qRT-PCR) on Abi Prism 7900 (Applied Biosystems). The JUM2 cell line was used as a calibrator for relative quantification of mRNA using the σσCt approach and Gus has been used as housekeeping gene. The cut-off value of Met mRNA expression/percentage calibrator was selected according to Receiver Operating Characteristic (ROC) analysis. ResultsmRNA expression of Met was higher in CD138+ cells than in CD138- cells (median 103, range 2 – 586 vs median 31, range 0-243 respectively; p=0.002). Met mRNA expression was significantly higher in CD138+ cells of patients with suboptimal response (partial response PR, stable disease SD, progression disease PD), compared with patients achieving complete response (CR) or very good partial remission (VGPR) (median 140 range 27-586 vs median 36.5 range 1-163 respectively; p=0.0001) (FIG 1A). With a median follow-up of 18 months, the 2-year PFS was 54% in patients with high Met mRNA expression and 87% in patients with low Met mRNA expression (p=0.007, FIG 1B); 2-year OS was 72% in patients with high Met mRNA expression and 95% in patients with low Met mRNA expression (p=0.047, FIG 1C). Patients with high levels of Met mRNA displayed albumin and beta-2-microglobuline levels similar to that observed in patients with low levels of Met mRNA. The BM plasma cells infiltration was also comparable (median 61% vs 65%) within the two groups. A similar percentage of patients in both groups showed the following cytogenetic abnormalities: t(4;14), t(14;16), del17 and del13. Interestingly, the percentage of patients carrying t(11;14) was higher in the group of patients with low Met mRNA levels in comparison with patients with high Met mRNA levels (28% vs 6%). mRNA expression of HGF was similar in responders and non responders patients and did not predict PFS and OS. Conclusion1) mRNA level of Met on CD 138+ cells is a biological marker predictive of response; 2) high level of Met mRNA at diagnosis delineates significantly inferior progression-free and overall survival in MM patients treated with both bortezomib and lenalidomide based regimens. [Display omitted] DisclosuresPatriarca:Celgene: Honoraria. Caravita:Celgene: Consultancy. Ladetto:Celgene: Research Funding; Janssen-Cilag: Research Funding. Boccadoro:Celgene: Consultancy, advisory Committees, Research Funding; Janssen-Cilag: Consultancy, advisory Committees, Research Funding; Pharmion: Consultancy, advisory Committees, Research Funding. Palumbo:Celgene: Honoraria; Janssen-Cilag: Honoraria.

Alteration of the Axial Met Ligand to Electron Acceptor A0 in Photosystem I: An Investigation of Electron Transfer at Different Temperatures by Multifrequency Time-Resolved and CW EPR

The ambient temperature and low-temperature electron transfer properties of Photosystem I (PS I) from the M688NPsaA and M668NPsaB mutant strains of the cyanobacterium Synechocystis sp. PCC 6803 are studied using transient electron paramagnetic resonance (EPR) and continuous-wave (CW) EPR. The two mutations are expected to alter the midpoint potentials of, and the reorganization energies around, the primary electron chlorophyll acceptors A0A and A0B, which should lead to a change in the yield and/or rate of electron transfer to the phylloquinone acceptors A1A and A1B, respectively. At ambient temperature it is known that both quinone acceptors are active in electron transfer. At low temperature there are at least two fractions that undergo either reversible or irreversible electron transfer. The EPR data of the two PS I variants are used to investigate the relationship between these low-temperature fractions and the ambient temperature electron transfer pathway. The results show that mutation in the PsaA-branch increases the rate of \( {\text{A}}_{{1{\text{A}}}}^{. - } \) to FX electron transfer at ambient temperature, while the corresponding mutation in the PsaB-branch has no effect on the electron transfer rate observable by transient EPR. An analysis of the complete time/field datasets from both variants suggests that the yield of electron transfer in the branch carrying the mutation is reduced. The mutations have no effect on the low-temperature CW EPR spectra of the iron–sulfur clusters if the samples are frozen under illumination but they both cause a decrease in the yield of reduced FA and FB if the samples are frozen in the dark and then illuminated. The PsaA-branch mutation greatly reduces the intensity and changes the polarization pattern of the radical pair \( {\text{P}}_{700}^{ + } {\text{A}}_{1}^{. - } \). Possible causes of the changes in the polarization pattern are discussed and it is suggested that the mutations introduce structural heterogeneity in the vicinity of the A0 binding site. No clear correlation between the yield of electron transfer in a particular branch and the yield of stable charge separation is found.

Hepatocyte growth factor as inducer of gefitinib resistance in non-small cell lung cancer harboring EGFR activating mutations

e19034 Background: Non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR)-activating mutations (EGFRmu) responds favorably to the EGFR tyrosine kinase inhibitors (EGFR-TKIs), gefitinib or erlotinib. However, 25–30% of patients with EGFRmu show intrinsic resistance, and the responders invariably acquire resistance to EGFR-TKIs. Here, we examined the role of hepatocyte growth factor (HGF), a specific ligand of MET, in gefitinib resistance of NSCLC with EGFRmu. Methods: NSCLC cells, PC-9 and HCC827, with EGFRmu, but not T790M second mutation in EGFR or MET amplification, were used in this study. Sensitivity to gefitinib was determined by MTT assay. Phosphorylation of MET, EGFR, ERBB3, and the PI3K/Akt pathway were examined by Western blotting. HGF expression in 20 tumors from 16 NSCLC patients with EGFRmu who were treated with gefitinib was determined by immunohistochemistry. Results: HGF induced resistance to gefitinib by restoring Akt phosphorylation independently of ErbB3. Specific down-regulation of MET, but not ErbB3, reversed gefitinib resistance and Akt phosphorylation induced by HGF. Importantly, high levels of immunoreactivity for HGF were detected in cancer cells of 3 of 3 intrinsic resistant tumors and in one of 2 tumors with acquired resistance without T790M second mutation or MET amplification. Conclusions: The findings indicate that HGF-mediated MET activation is a novel mechanism of intrinsic and acquired gefitinib-resistance in NSCLC with EGFRmu. Therefore, inhibition of HGF-MET signaling may be considerable strategy for more successful treatment with EGFR-TKIs. [Table: see text]

Metal-binding loop length and not sequence dictates structure

The C-terminal copper-binding loop in the β-barrel fold of the cupredoxin azurin has been replaced with a range of sequences containing alanine, glycine, and valine residues to assess the importance of amino acid composition and the length of this region. The introduction of 2 and 4 alanines between the coordinating Cys, His, and Met results in loop structures matching those in naturally occurring proteins with the same loop lengths. A loop with 4 alanines between the Cys and His and 3 between the His and Met ligands has a structure identical to that of the WT protein, whose loop is the same length. Loop structure is dictated by length and not sequence allowing the properties of the main surface patch for interactions with partners, to which the loop is a major contributor, to be optimized. Loops with 2 amino acids between the ligands using glycine, alanine, and valine residues have been compared. An empirical relationship is found between copper site protection by the loop and reduction potential. A loop adorned with 4 methyl groups is sufficient to protect the copper ion, enabling most sequences to adequately perform this task. The mutant with 3 alanine residues between the ligands forms a strand-swapped dimer in the crystal structure, an arrangement that has not, to our knowledge, been seen previously for this family of proteins. Cupredoxins function as redox shuttles and are required to be monomeric; therefore, none have evolved with a metal-binding loop of this length.

Active site loop dictates the thermodynamics of reduction and ligand protonation in cupredoxins

The thermodynamics of reduction and His ligand protonation have been determined for a range of loop-contraction variants of the electron transferring type 1 copper protein azurin (AZ). For AZPC, in which the native C-terminal loop containing the Cys, His and Met ligands has been replaced with the shorter sequence from plastocyanin (PC) and AZAMI, in which the even shorter amicyanin (AMI) loop has been inserted, the thermodynamics of reduction match those of the protein whose loop has been introduced which are different to the values for AZ. The enthalpic contribution to His ligand protonation, which is not observed in AZ, is similar in AZAMI and AMI. The thermodynamics of this process in AZPC are more dissimilar to those for PC. In the case of AZAMI-F, a variant possessing the (non natural) minimal loop that can bind a type 1 copper site, the reduction thermodynamics are intermediate between those of AZPC and AZAMI, whilst the thermodynamic data for His ligand protonation are very similar to those for AMI. The results for AZAMI and AZPC are primarily due to protein based enthalpic effects related to the interaction of the metal with permanent protein dipoles from the loop, and to the decreased loop length which favors His ligand protonation in the cuprous proteins. Entropic factors related to loop flexibility have little influence because of constraints imposed by metal coordination and the fact that the introduced loops pack well against the AZ scaffold. Thus, the host scaffold in general plays a minor thermodynamic role in both processes, although for AZAMI-F differences in the first and second coordination spheres influence the thermodynamics of reduction.

MET, HGF, EGFR, and PXN Gene Copy Number in Lung Cancer Using DNA Extracts from FFPE Archival Samples and Prognostic Significance

Gene copy number analysis for some of the important molecules in lung tumorogenesis, such as MET, hepatocyte growth factor [(HGF), ligand for MET), epidermal growth factor receptor (EGFR), and paxillin (PXN), is likely to determine both the type of treatment and prognosis. Formalin-fixed paraffin-embedded (FFPE) archival tumor tissue samples are an excellent source for determining key molecular changes in the OncoGenome; however, existing extraction procedures yield relatively poor quality genomic DNA fragments. Although FISH is the method of choice for determining amplification of a gene, a more rapid quantitative poly-merase chain reaction (qPCR) technique to determine gene copy number can be used when reasonably good quality genomic DNA is available. We report here a relatively rapid method based on microwave/chelex-100 treatment that gives rise to genomic DNA fragments ranging from 1 to 12 Kb and beyond, thereby attesting to its superior quality. Genomic PCR for beta-globin gene gave reliable and reproducible results. The number of steps for extracting the DNA was kept to a minimum, and instead of precipitating the DNA, we preserved the genomic DNA extracts so as to prevent a loss in DNA yield. We found the extracts to be stable and amenable to qPCR and mutational analysis. Using lung adenocarcinoma FFPE samples and cell lines derived from lung adenocarcinomas, we demonstrated that the gene copy number for MET in lung adenocarcinoma tissue samples was preferentially increased over EGFR, HGF, and PXN and that it positively correlated with a better prognosis. In contrast, the genomic DNA extracted from 25 NSCLC cell lines gave a relatively higher gene copy number for all four genes evaluated. Our results indicate that the microwave/chelex-100-based method yields good-quality genomic DNA extracts that can be used for complex DNA analysis, such as determination of gene copy number. In addition, our data demonstrated that the adenocarcinoma cell lines potentially evolved under ex vivo conditions, and therefore, in genetic studies it is imperative to use primary tumors for generalized conclusions about lung tumors.