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The hepatocyte growth factor (HGF)/c-Met signaling pathway is involved in the progression of several cancers and associated with increased tumor invasion and metastatic potential. We determined previously that the polyphenol epigallocatechin-3-gallate inhibited HGF-induced c-Met phosphorylation in a variety of tumor cell lines in part by disrupting lipid rafts. Fatty acid synthase (FASN) is implicated in cancer progression and may regulate lipid raft function. We therefore examined the effects of luteolin, a potent FASN inhibitor, on c-Met signaling. Luteolin blocked HGF-induced c-Met phosphorylation and scattering of DU145 prostate cancer cells, but inhibition required at least a 4 h preincubation time. Western blot analysis indicated that inhibition of HGF-induced scattering by luteolin occurred coincident with reduction of total c-Met protein in DU145 cells. In addition, luteolin-induced c-Met down-regulation was mimicked by a pharmacologic inhibitor of FASN, C75, or short hairpin RNA knockdown of FASN. Consistent with a role for FASN, loss of c-Met in cells treated with C75 or luteolin was prevented by exogenous addition of palmitate. Luteolin-induced loss of c-Met primarily occurred at a post-transcriptional level and involved cell surface internalization but did not involve translation inhibition, nor was it dependent on the activity of the 26S proteosome or acidic lysosomes. Taken together, our study shows a novel connection between FASN activity and c-Met protein expression and suggests that luteolin could act as a novel HGF/c-Met inhibitor by reducing expression of this receptor. [Mol Cancer Ther 2009;8(1):214–24]

Supplementary Data from The Green Tea Polyphenol EGCG Potentiates the Antiproliferative Activity of c-Met and Epidermal Growth Factor Receptor Inhibitors in Non–small Cell Lung Cancer Cells

Supplementary Fig. S1 from Inhibition of fatty acid synthase by luteolin post-transcriptionally down-regulates c-Met expression independent of proteosomal/lysosomal degradation

Purpose: Activation of the c-Met and epidermal growth factor receptors (EGFR) promotes the growth and survival of non–small cell lung cancer (NSCLC). Specific receptor antagonists have shown efficacy in the clinic, but tumors often become resistant to these therapies. We investigated the ability of (-)-epigallocatechin-3-gallate (EGCG) to inhibit cell proliferation, and c-Met receptor and EGFR kinase activation in several NSCLC cell lines.Experimental Design: NSCLC cell lines with variable sensitivity to the EGFR antagonist erlotinib were studied. Cell growth was evaluated using proliferation and colony formation assays. Kinase activation was assessed via Western blot analysis. Experiments were conducted with EGCG, the EGFR antagonist erlotinib, and the c-Met inhibitor SU11274. The antagonists were also tested in a xenograft model using SCID mice.Results: EGCG inhibited cell proliferation in erlotinib-sensitive and -resistant cell lines, including those with c-Met overexpression, and acquired resistance to erlotinib. The combination of erlotinib and EGCG resulted in greater inhibition of cell proliferation and colony formation than either agent alone. EGCG also completely inhibited ligand-induced c-Met phosphorylation and partially inhibited EGFR phosphorylation. The triple combination of EGCG/erlotinib/SU11274 resulted in a greater inhibition of proliferation than EGCG with erlotinib. Finally, the combination of EGCG and erlotinib significantly slowed the growth rate of H460 xenografts.Conclusion: EGCG is a potent inhibitor of cell proliferation, independent of EGFR inhibition, in several NSCLC cell lines, including those resistant to both EGFR kinase inhibitors and those overexpressing c-Met. Therefore, EGCG might be a useful agent to study as an adjunct to other anticancer agents.

Cancer is a multistep process that requires cells to respond appropriately to the tumor microenvironment, both in early proliferative stages and in later invasive disease. Arl8b is a lysosome localized Arf-like GTPase that controls the spatial distribution of lysosomes via recruitment of kinesin motors. Common features of the tumor microenvironment such as acidic extracellular pH and various growth factors stimulate lysosome trafficking to the cell periphery (anterograde), which is critical for tumor invasion by facilitating the release of lysosomal proteases to promote matrix remodeling. Herein we report for the first time that Arl8b regulates anterograde lysosome trafficking in response to hepatocyte growth factor, epidermal growth factor, and acidic extracellular pH. Depletion of Arl8b results in juxtanuclear lysosome aggregation, and this effect corresponds with both diminished invasive growth and proteolytic extracellular matrix degradation in a three-dimensional model of prostate cancer. Strikingly, we found that depletion of Arl8b abolishes the ability of prostate cancer cells to establish subcutaneous xenografts in mice. We present evidence that Arl8b facilitates lipid hydrolysis to maintain efficient metabolism for a proliferative capacity in low nutrient environments, suggesting a likely explanation for the complete inability of Arl8b-depleted tumor cells to grow in vivo. In conclusion, we have identified two mechanisms by which Arl8b regulates cancer progression: 1) through lysosome positioning and protease release leading to an invasive phenotype and 2) through control of lipid metabolism to support cellular proliferation. These novel roles highlight that Arl8b is a potential target for the development of novel anti-cancer therapeutics.

The tumor microenvironment, primarily composed of myofibroblasts, directly influences the progression of solid tumors. Through secretion of growth factors, extracellular matrix deposition, and contractile mechanotransduction, myofibroblasts, or cancer-associated fibroblasts (CAFs), support angiogenesis and cancer cell invasion and metastasis. The differentiation of fibroblasts to CAFs is primarily induced by TGF-β from cancer cells. To discover agents capable of blocking CAF differentiation, we developed a high content immunofluorescence-based assay to screen repurposed chemical libraries utilizing fibronectin expression as an initial CAF marker. Screening of the Prestwick chemical library and NIH Clinical Collection repurposed drug library, totaling over 1700 compounds, identified cardiac glycosides as particularly potent CAF blocking agents. Cardiac glycosides are traditionally used to regulate intracellular calcium by inhibiting the Na+/K+ ATPase to control cardiac contractility. Herein, we report that multiple cardiac glycoside compounds, including digoxin, are able to inhibit TGF-β-induced fibronectin expression at low nanomolar concentrations without undesirable cell toxicity. We found this inhibition to hold true for multiple fibroblast cell lines. Using real-time qPCR, we determined that digoxin prevented induction of multiple CAF markers. Furthermore, we report that digoxin is able to prevent TGF-β-induced fibroblast contraction of extracellular matrix, a major phenotypic consequence of CAF differentiation. Assessing the mechanism of inhibition, we found digoxin reduced SMAD promoter activity downstream of TGF-β, and we provide data that the effect is through inhibition of its known target, the Na+/K+ ATPase. These findings support a critical role for calcium signaling during CAF differentiation and highlight a novel, repurposable modality for cancer therapy.

// Alana L. Gray 1, 2 , David T. Coleman 1, 2 , Runhua Shi 1, 2 , James A. Cardelli 1, 2 1 Louisiana State University Health Sciences Center–Shreveport, Shreveport, LA, USA 2 Feist-Weiller Cancer Center, Shreveport, LA, USA Correspondence to: Alana L. Gray, email: agray@lsuhsc.edu Keywords: MCT1, HGF, EGF, cancer, motility Received: January 26, 2016 Accepted: March 31, 2016 Published: April 26, 2016 ABSTRACT Tumor progression to metastatic disease contributes to the vast majority of incurable cancer. Understanding the processes leading to advanced stage cancer is important for the development of future therapeutic strategies. Here, we establish a connection between tumor cell migration, a prerequisite to metastasis, and monocarboxylate transporter 1 (MCT1). MCT1 transporter activity is known to regulate aspects of tumor progression and, as such, is a clinically relevant target for treating cancer. Knockdown of MCT1 expression caused decreased hepatocyte growth factor (HGF)-induced as well as epidermal growth factor (EGF)-induced tumor cell scattering and wound healing. Western blot analysis suggested that MCT1 knockdown (KD) hinders signaling through the HGF receptor (c-Met) but not the EGF receptor. Exogenous, membrane-permeable MCT1 substrates were not able to rescue motility in MCT1 KD cells, nor was pharmacologic inhibition of MCT1 able to recapitulate decreased cell motility as seen with MCT1 KD cells, indicating transporter activity of MCT1 was dispensable for EGF- and HGF-induced motility. These results indicate MCT1 expression, independent of transporter activity, is required for growth factor-induced tumor cell motility. The findings presented herein suggest a novel function for MCT1 in tumor progression independent of its role as a monocarboxylate transporter.

Background Even though 8%‐25% of most populations studied globally are labeled as penicillin allergic, most diagnoses of penicillin allergy are made in childhood and relate to events that are either not allergic in nature, are low risk for immediate hypersensitivity, or are a potential true allergy that has waned over time. Penicillin allergy labels directly impact antimicrobial stewardship by leading to use of less effective and broader spectrum antimicrobials and are associated with antimicrobial resistance. They may also delay appropriate antimicrobial therapy and lead to increased risk of specific adverse healthcare outcomes. Operationalizing penicillin allergy de‐labeling into a new arm of antimicrobial stewardship programs (ASPs) has become an increasing global focus. Methods We performed an evidence‐based narrative review of the literature of penicillin allergy label carriage, the adverse effects of penicillin allergy labels, and current approaches and barriers to penicillin allergy de‐labeling. Over the period 1928‐2018 in Pubmed and Medline, search terms used included “penicillin allergy” or “penicillin hypersensitivity” alone or in combination with “adverse events,” “testing,” “evaluation,” “effects,” “label,” “de‐labeling,” “prick or epicutaneous,” and “intradermal” skin testing, “oral challenge or provocation,” “cross‐reactivity,” and “antimicrobial stewardship”. Results Penicillin allergy labels are highly prevalent, largely inaccurate and their carriage may lead to unnecessary treatment and inferior outcomes with alternative agents as well as adverse public health outcomes such as antibiotic resistance. Conclusions Operationalizing penicillin allergy de‐labeling as an aspect of ASP has become an increasing global focus. There is a need for validated approaches that optimally combine the use of history and ingestion challenge with or without proceeding formal skin testing to tackle penicillin allergy efficiently within complex healthcare systems. At the same time, there is great promise for penicillin allergy evaluation and de‐labeling as an individual and public health strategy to reduce adverse healthcare outcomes, improve antimicrobial stewardship, and decrease healthcare costs.

// Alana L. Gray 1, 2 , David T. Coleman 1, 2 , Reneau F. Castore 1, 2 , Mohamed M. Mohyeldin 3 , Khalid A. El Sayed 3 , James A. Cardelli 1, 2 1 Louisiana State University Health Sciences Center - Shreveport, Shreveport, LA, USA 2 Feist-Weiller Cancer Center, Shreveport, LA, USA 3 University of Louisiana - Monroe, Monroe, LA, USA Correspondence to: James A. Cardelli, e-mail: jcarde@lsuhsc.edu Keywords: c-Met, isothiocyanatostilbenes, DIDS, H2DIDS Received: August 05, 2015 Accepted: September 17, 2015 Published: October 31, 2015 ABSTRACT The hepatocyte growth factor receptor (HGFR or c-Met) is a driver of multiple cancer subtypes. While there are several c-Met inhibitors in development, few have been approved for clinical use, warranting the need for continued research and development of c-Met targeting therapeutic modalities. The research presented here demonstrates a particular class of compounds known as isothiocyanatostilbenes can act as c-Met inhibitors in multiple cancer cell lines. Specifically, we found that 4,4′-Diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and 4,4′-Diisothiocyanatodihydrostilbene-2,2′-disulfonic acid (H2DIDS) had c-Met inhibitory effective doses in the low micromolar range while 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS) and 4,4′-dinitrostilbene-2, 2′-disulfonic acid (DNDS) exhibited IC 50 s 100 to 1000 fold higher. These compounds displayed much greater selectivity for inhibiting c-Met activation compared to similar receptor tyrosine kinases. In addition, DIDS and H2DIDS reduced hepatocyte growth factor (HGF)-induced, but not epidermal growth factor (EGF)-induced, cell scattering, wound healing, and 3-dimensional (3D) proliferation of tumor cell spheroids. In-cell and cell-free assays suggested that DIDS and H2DIDS can inhibit and reverse c-Met phosphorylation, similar to SU11274. Additional data demonstrated that DIDS is tolerable in vivo . These data provide preliminary support for future studies examining DIDS, H2DIDS, and derivatives as potential c-Met therapeutics.

Objective— Macrophage proinflammatory responses induced by modified low-density lipoproteins (modLDL) contribute to atherosclerotic progression. How modLDL causes macrophages to become proinflammatory is still enigmatic. Macrophage foam cell formation induced by modLDL requires glycerolipid synthesis. Lipin-1, a key enzyme in the glycerolipid synthesis pathway, contributes to modLDL-elicited macrophage proinflammatory responses in vitro. The objective of this study was to determine whether macrophage-associated lipin-1 contributes to atherogenesis and to assess its role in modLDL-mediated signaling in macrophages. Approach and Results— We developed mice lacking lipin-1 in myeloid-derived cells and used adeno-associated viral vector 8 expressing the gain-of-function mutation of mouse proprotein convertase subtilisin/kexin type 9 (adeno-associated viral vector 8-proprotein convertase subtilisin/kexin type 9) to induce hypercholesterolemia and plaque formation. Mice lacking myeloid-associated lipin-1 had reduced atherosclerotic burden compared with control mice despite similar plasma lipid levels. Stimulation of bone marrow–derived macrophages with modLDL activated a persistent protein kinase Cα/βII–extracellular receptor kinase1/2–jun proto-oncogene signaling cascade that contributed to macrophage proinflammatory responses that was dependent on lipin-1 enzymatic activity. Conclusions— Our data demonstrate that macrophage-associated lipin-1 is atherogenic, likely through persistent activation of a protein kinase Cα/βII–extracellular receptor kinase1/2–jun proto-oncogene signaling cascade that contributes to foam cell proinflammatory responses. Taken together, these results suggest that modLDL-induced foam cell formation and modLDL-induced macrophage proinflammatory responses are not independent consequences of modLDL stimulation but rather are both directly influenced by enhanced lipid synthesis.

Modified low density lipoproteins (modLDL) elicit macrophage generation into foam cells that release pro-inflammatory mediators driving atherosclerotic lesion progression causing cardiovascular disease. The molecular mechanisms that elicit foam cell inflammatory responses have not yet been fully elucidated. The lipid-laden phenotype that is characteristic of macrophage foam cells is due to lipid droplet biogenesis in response to excess cholesterol. Lipid droplet biogenesis is a process that is thought to be symptomatic of, but not drive atherosclerosis. Lipid droplet biogenesis requires glycerolipid synthesis, during which, lipin-1 converts phosphatidate into diglyceride as the penultimate step of lipid droplet generation. We had previously demonstrated lipin-1 is also required for modLDL-elicited pro-inflammatory response from macrophages. We hypothesized that modLDL elicits chronic diglyceride generation, via lipin-1 enzymatic activity, that activates signaling cascades responsible for foam cell pro-inflammatory responses. To test our hypotheses we stimulated wild type and lipin-1 depleted bone marrow-derived macrophages (BMDMs) with oxidized LDLs (oxLDLs). Stimulation of wild type BMDMs resulted in chronic activation of the signaling kinases PKCα/βII, ERK1/2 and the AP-1 transcription factor subunit cJun (up to 48 hours after stimulation). This pathway was not observed to be active in BMDMs depleted of lipin-1 either genetically or with siRNA. The pharmacological inhibition of lipin-1, PKCα/βII, ERK1/2 strongly suggest lipin-1- PKCα/βII-ERK1/2-cJun represents a signaling axis. Finally, each of these proteins were required for oxLDL-elicited pro-inflammatory responses by macrophages. These results suggest that augmented glycerolipid synthesis in macrophages due to modLDL stimulation is not just symptomatic of atherosclerosis but promote inflammatory responses that drive lesion progression

The hepatocyte growth factor (HGF)/c-Met signaling pathway is involved in the progression of several cancers and associated with increased tumor invasion and metastatic potential. We determined previously that the polyphenol epigallocatechin-3-gallate inhibited HGF-induced c-Met phosphorylation in a variety of tumor cell lines in part by disrupting lipid rafts. Fatty acid synthase (FASN) is implicated in cancer progression and may regulate lipid raft function. We therefore examined the effects of luteolin, a potent FASN inhibitor, on c-Met signaling. Luteolin blocked HGF-induced c-Met phosphorylation and scattering of DU145 prostate cancer cells, but inhibition required at least a 4 h preincubation time. Western blot analysis indicated that inhibition of HGF-induced scattering by luteolin occurred coincident with reduction of total c-Met protein in DU145 cells. In addition, luteolin-induced c-Met down-regulation was mimicked by a pharmacologic inhibitor of FASN, C75, or short hairpin RNA knockdown of FASN. Consistent with a role for FASN, loss of c-Met in cells treated with C75 or luteolin was prevented by exogenous addition of palmitate. Luteolin-induced loss of c-Met primarily occurred at a post-transcriptional level and involved cell surface internalization but did not involve translation inhibition, nor was it dependent on the activity of the 26S proteosome or acidic lysosomes. Taken together, our study shows a novel connection between FASN activity and c-Met protein expression and suggests that luteolin could act as a novel HGF/c-Met inhibitor by reducing expression of this receptor. [Mol Cancer Ther 2009;8(1):214–24]

c-Met is a receptor tyrosine kinase whose activity can promote both mitogenic and motogenic phenotypes involved in tissue development and cancer progression. Herein, we report the first evidence that c-Met is palmitoylated and that palmitoylation facilitates its trafficking and stability. Inhibition of palmitoylation reduced the expression of c-Met in multiple cancer cell lines post-transcriptionally. Using surface biotinylation, confocal microscopy, and metabolic labeling we determined that inhibition of palmitoylation reduces the stability of newly synthesized c-Met and causes accumulation at the Golgi. Acyl-biotin exchange and click chemistry-based palmitate labeling indicated the c-Met β-chain is palmitoylated, and site-directed mutagenesis revealed two likely cysteine palmitoylation sites. Moreover, by monitoring palmitoylation kinetics during the biosynthesis and trafficking of c-Met, we revealed that stable palmitoylation occurs in the endoplasmic reticulum prior to cleavage of the 170 kDa c-Met precursor to the mature 140 kDa form. Our data suggest palmitoylation is required for egress from the Golgi for transport to the plasma membrane. These findings introduce palmitoylation as a critical modification of c-Met, providing a novel therapeutic target for c-Met-driven cancers.

Hany I Sakr,1–3 David T Coleman,3,4 James A Cardelli,3,4 J Michael Mathis,2,3,5 1Department of Cellular Biology and Anatomy, 2Gene Therapy Program, 3Feist-Weiller Cancer Center, 4Department of Microbiology and Immunology, LSU Health Shreveport, Shreveport, 5Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, USA Abstract: The cMet receptor is a homodimer with tyrosine kinase activity. Upon stimulation with its ligand, hepatocyte growth factor (HGF), the receptor mediates wide physiologic actions. The HGF-cMet signaling pathway is dysregulated in many cancers, which makes cMet an important target for novel therapeutic interventions. Oncolytic adenoviruses (Ads) have been used for the past three decades as a promising therapeutic approach for a wide array of neoplastic diseases. To date, achieving cancer-specific replication of oncolytic Ads has been accomplished by either viral genome deletions or by incorporating tumor selective promoters. To achieve novel specificity of oncolytic Ad infection of cancer cells that overexpress cMet, we inserted the HGF NK2 sequence, corresponding to a competitive antagonist of HGF binding to the cMet receptor, into the Ad serotype 5 (Ad5) fiber gene. The resulting vector, Ad5-pIX-RFP-FF/NK2, was rescued, amplified in HEK293 cells, and characterized. Binding specificity and viral infectivity were tested in various cancer cell lines that express varying levels of cMet and hCAR (the Ad5 receptor). We found that Ad5-pIX-RFP-FF/NK2 demonstrated binding specificity to the cMet receptor. In addition, there was enhanced viral infectivity and virus replication compared with a non-targeted Ad vector. Although NK2 weakly induces cMet receptor activation, our results showed no receptor phosphorylation in the context of an oncolytic Ad virus. In summary, these results suggest that an oncolytic Ad retargeted to the cMet receptor is a promising vector for developing a novel cancer therapeutic agent. Keywords: bacteriophage T4 fibritin, fiber protein, gene therapy, hCAR, hepatocyte growth factor, NK2 isoform, oncolytic virotherapy, pIX protein, red fluorescent protein

Curcumin has been shown to mitigate cancer phenotypes such as invasive migration, proliferation, and survival by disrupting numerous signaling pathways. Our previous studies showed that curcumin inhibits integrin β4 (ITG β4)-dependent migration by blocking interaction of this integrin with growth factor receptors in lipid rafts. In the current study, we investigated the possibility that curcumin inhibits ITG β4 palmitoylation, a post-translational modification required for its lipid raft localization and signaling activity. We found that the levels of ITG β4 palmitoylation correlated with the invasive potential of breast cancer cells, and that curcumin effectively reduced the levels of ITG β4 palmitoylation in invasive breast cancer cells. Through studies of ITG β4 palmitoylation kinetics, we concluded curcumin suppressed palmitoylation independent of growth factor-induced phosphorylation of key ITG β4 Ser and Tyr residues. Rather, curcumin blocked autoacylation of the palmitoyl acyltransferase DHHC3 that is responsible for ITG β4 palmitoylation. Moreover, these data reveal that curcumin is able to prevent the palmitoylation of a subset of proteins, but not indiscriminately bind to and block all cysteines from modifications. Our studies reveal a novel paradigm for curcumin to account for much of its biological activity, and specifically, how it is able to suppress the signaling function of ITG β4 in breast cancer cells.

Invasion and subsequent metastasis is the major cause of death from most cancers including prostate cancer. Herein we report on the potential tumor suppressive properties of Rab7, a GTPase that regulates trafficking of lysosomes. The movement of lysosomes to the cell surface in response to environmental cues increases the secretion of proteinases and cell invasion. We determined that Troglitazone and other members of the Thiazolidinedione family inhibit cell-surface directed lysosome trafficking and cathepsin B secretion through a Rab7-dependent mechanism. Moreover, Rab7 shRNA expressing cells were found to be more invasive in vitro and in vivo. Increased invasiveness was accompanied by elevated expression of the c-Met receptor and prolonged downstream signaling, thereby supporting a role for Rab7 as a mediator of signaling down-regulation. Taken together, these results suggested that Rab7 acts as a negative regulator of prostate tumor growth and invasion, providing further evidence for its potential as a tumor suppressor.

Prostate cancer (PCa) is the most common noncutaneous neoplasia diagnosed in men and represents roughly 10% of cancer-associated mortalities. The lethal phenotype of PCa is primarily characterized by progression of tumor cells to castration-resistance and metastasis. Organ-confined PCa is curable with surgery and/or radiation therapy, however, as the disease becomes metastatic, the likelihood of survival becomes minimal. Key influences in the progression to metastasis are growth factor receptors including the receptor tyrosine kinase c-Met. During this research period, I have acquired convincing data that the c-Met receptor tyrosine kinase is palmitoylated and that this palmitoylation regulates its stability. Inhibition of palmitoylation reduces the expression of c-Met in prostate cancer cell lines. This protein loss occurs post-transcriptionally and is associated with accumulation of c-Met in Golgi compartments. Using inhibitors to a number of internalization pathways, as well as surface biotinylation studies and confocal microscopy, we determined that inhibition of palmitoylation reduces the stability of newly synthesized c-Met as opposed to inducing internalization and degradation. Moreover, both an acyl-biotin exchange technique and a click-chemistry based palmitate-labeling protocol suggest c-Met itself is palmitoylated. Observing palmitoylation kinetics has provided evidence that c-Met is palmitoylated in the Golgi prior to cleavage of the c- Met precursor. Currently, mass spectrometry analysis is being performed to identify the specific cysteine residue/s that are palmitoylated to aid future studies. Taken together, these findings suggest inhibition of palmitoylation could be a novel target for treating prostate cancer driven by oncogenic c-Met.

The Met tyrosine kinase receptor is the only known receptor for hepatocyte growth factor (HGF). Downstream Met signaling is essential for embryonic development; however, aberrant Met signaling promotes tumor progression by facilitating cell proliferation, survival, migration, invasion, and metastasis. Tumor cell invasion is considered an important step in distant metastatic foci formation. Several recent reviews have focused on the pleiotropic effects of Met signaling in both tumor cells and in the surrounding stromal cells. This review will summarize the currently described mechanisms driving Met induced tumor cell progression and invasion, the role played by cells in the tumor stroma, and therapeutic approaches to block receptor activity. In addition, this review will also highlight two new areas of development: 1) attenuation of Met signaling via multiple mechanisms of action targeting tumor cells and cells in the surrounding stroma using plant-derived polyphenols and 2) the induction by HGF of atypical lysosome trafficking, leading to increased protease secretion and tumor cell invasion. These new areas of research will help to uncover novel therapeutic targets to block the HGF/Met signaling axis to slow cancer progression.

The HGF/c-Met pathway is an important regulator of signaling pathways responsible for invasion and metastasis of most human cancers, including prostate cancer. Exposure of DU145 prostate tumor cells to HGF stimulates the PI3-kinase and MAPK pathways, leading to increased scattering, motility, and invasion, which was prevented by the addition of EGCG. EGCG acted at the level of preventing phosphorylation of tyrosines 1234/1235 in the kinase domain of the c-Met receptor without effecting dimerization. HGF-induced changes were independent of the formation of reactive oxygen species, suggesting that EGCG functioned independent of its antioxidant ability. ECG, another tea polyphenol, was as effective as EGCG, while EGC and EC were less effective. EGCG added up to 4 h after the addition of HGF still blocked cell scattering and reduced the HGF-induced phosphorylation of c-Met, Akt, and Erk, suggesting that EGCG could act both by preventing activation of c-Met by HGF and by attenuating the activity of pathways already induced by HGF. HGF did not activate the MAPK and PI3-K pathways in cells treated with methyl-β-cyclodextrin (mCD) to remove cholesterol. Furthermore, subcellular fractionation approaches demonstrated that only phosphorylated c-Met accumulated in Triton X-100 membrane insoluble fractions, supporting a role for lipid rafts in regulating c-Met signaling. Finally, EGCG treatment inhibited DiIC16 incorporation into membrane lipid ordered domains, and cholesterol partially inhibited the EGCG effects on signaling. Together, these results suggest that green tea polyphenols with the R1 galloyl group prevent activation of the c-Met receptor by altering the structure or function of lipid rafts. © 2010 Wiley-Liss, Inc.

Purpose: Activation of the c-Met and epidermal growth factor receptors (EGFR) promotes the growth and survival of non–small cell lung cancer (NSCLC). Specific receptor antagonists have shown efficacy in the clinic, but tumors often become resistant to these therapies. We investigated the ability of (-)-epigallocatechin-3-gallate (EGCG) to inhibit cell proliferation, and c-Met receptor and EGFR kinase activation in several NSCLC cell lines. Experimental Design: NSCLC cell lines with variable sensitivity to the EGFR antagonist erlotinib were studied. Cell growth was evaluated using proliferation and colony formation assays. Kinase activation was assessed via Western blot analysis. Experiments were conducted with EGCG, the EGFR antagonist erlotinib, and the c-Met inhibitor SU11274. The antagonists were also tested in a xenograft model using SCID mice. Results: EGCG inhibited cell proliferation in erlotinib-sensitive and -resistant cell lines, including those with c-Met overexpression, and acquired resistance to erlotinib. The combination of erlotinib and EGCG resulted in greater inhibition of cell proliferation and colony formation than either agent alone. EGCG also completely inhibited ligand-induced c-Met phosphorylation and partially inhibited EGFR phosphorylation. The triple combination of EGCG/erlotinib/SU11274 resulted in a greater inhibition of proliferation than EGCG with erlotinib. Finally, the combination of EGCG and erlotinib significantly slowed the growth rate of H460 xenografts. Conclusion: EGCG is a potent inhibitor of cell proliferation, independent of EGFR inhibition, in several NSCLC cell lines, including those resistant to both EGFR kinase inhibitors and those overexpressing c-Met. Therefore, EGCG might be a useful agent to study as an adjunct to other anticancer agents.

The influence of growth factor receptors on cancer progression as both early promoters as well as drivers of late-stage invasion and metastasis has been thoroughly studied over the last several decades. Overexpression and activating mutations of several receptor tyrosine kinases (RTKs) are commonly detected in most cancer types. Despite this, there still remains a great deal not fully understood about RTK expression and regulation that may reveal novel strategies for therapeutic targeting. In particular, c-Met is an RTK whose downstream signal transduction can promote both mitogenic and motogenic phenotypes in cancer cells and whose expression is correlated with poor prognosis and resistance to therapy. c-Met is activated by either autocrine or paracrine ligand stimulation. Alternatively, receptor overexpression allows for ligand-independent dimerization and therefore constitutive activation. In addition, multiple reports have identified the enzyme fatty acid synthase (FASN) as being commonly overexpressed in prostate cancer, and that this aberrant expression is an early event that becomes more pronounced with aggressive androgen-independent and metastatic disease. FASN is the sole enzyme responsible for de novo synthesis of the 16-carbon saturated fatty acid palmitate. In cancer, de novo lipids are more selectively partitioned into lipid rafts as phospholipids as well as utilized for post-translational acyl-modifications of signaling proteins. Previous findings have led us to identify a novel mechanism by which FASN activity regulates c-Met expression. Our work has determined that inhibition or shRNA knockdown of FASN results in a post-translational downregulation of already synthesized c-Met protein. This downregulation is prevented by the addition of exogenous palmitate. Based on these findings we have subsequently acquired convincing data that the c-Met receptor tyrosine kinase is palmitoylated and that this palmitoylation regulates its stability. Inhibition of palmitoylation reduces the expression of c-Met in multiple cancer cell lines. This protein loss occurs post-transcriptionally and is associated with accumulation of c-Met in Golgi compartments. Using inhibitors to a number of internalization pathways, as well as surface biotinylation studies, confocal microscopy, and metabolic-ortholog labeling we determined that inhibition of palmitoylation reduces the stability of newly synthesized, c-Met as opposed to inducing internalization and degradation. Moreover, both an acyl-biotin exchange technique and a click-chemistry based palmitate-labeling protocol suggest c-Met itself is palmitoylated. Observing palmitoylation kinetics has provided evidence that c-Met is palmitoylated in the ER prior to cleavage of the 170kd c-Met precursor into its mature 140kd form. Taken together, these findings suggest inhibition of palmitoylation or FASN activity could be a novel target for preventing invasion and metastasis driven by c-Met overexpression. Citation Format: David T. Coleman, James A. Cardelli. c-Met requires palmitoylation for proper stability and trafficking in cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A83.

Prostate cancer (PCa) is the most common noncutaneous neoplasia diagnosed in men and represents roughly 10% of cancer-associated mortalities. The lethal phenotype of PCa is primarily characterized by progression of tumor cells to androgen-independence and metastasis. Organ-confined PCa is often curable with surgery and/or radiation therapy, however, as the disease becomes metastatic, the likelihood of survival becomes minimal. Key influences in the progression to metastasis are growth factor receptors including the receptor tyrosine kinase c-Met. Numerous bodies of evidence strongly link aberrant c-Met signaling with causation and/or progression of prostate cancer. Overexpression of c-Met can lead to activation in a ligand-independent manner and has been identified as a common mechanism of resistance to RTK-targeted therapy. In addition, multiple reports have identified the enzyme fatty acid synthase (FASN) as being commonly overexpressed in prostate cancer, and that this aberrant expression is an early event that becomes more pronounced with aggressive androgen-independent and metastatic disease. FASN is the sole enzyme responsible for de novo synthesis of the 16-carbon saturated fatty acid palmitate. In cancer, de novo lipids are more selectively partitioned into lipid rafts as phospholipids as well as utilized for post-translational acyl-modifications of signaling proteins. Previous findings have led us to identify a novel mechanism by which FASN activity regulates c-Met expression. Our work has determined that inhibition or shRNA knockdown of FASN results in a post-translational downregulation of already synthesized c-Met protein. This downregulation is prevented by the addition of exogenous palmitate. Here we provide evidence that inhibition of palmitoylation, using the palmitate analog 2-bromopalmitate, lowers total c-Met levels. This protein loss occurs post-transcriptionally and is associated with accumulation of c-Met in Golgi compartments. Using inhibitors to a number of internalization pathways, as well as surface biotinylation studies, our findings suggest the inhibition of palmitoylation reduces the stability of newly synthesized c-Met as opposed to inducing internalization and degradation. Moreover, both an acyl-biotin exchange technique and a click-chemistry based palmitate-labeling protocol have provided evidence suggesting c-Met itself is palmitoylated. From these data, we hypothesize that c-Met requires palmitoylation along a biosynthetic route to promote its stability and trafficking to the cell surface. In the absence of palmitoylation, c-Met accumulates within the Golgi and is disposed of through a yet to be determined degradation pathway. Our findings potentially reveal a novel mechanism for restricting the expression of c-Met as a means of preventing prostate cancer invasion and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1216. doi:1538-7445.AM2012-1216