Pyridine-pyrimidine amides that prevent HGF-induced epithelial scattering by two distinct mechanisms

Stimulation of cultured epithelial cells with scatter factor/hepatocyte growth factor (HGF) results in individual cells detaching and assuming a migratory and invasive phenotype. Epithelial scattering recapitulates cancer progression and studies have implicated HGF signaling as a driver of cancer metastasis. Inhibitors of HGF signaling have been proposed to act as anti-cancer agents. We previously screened a small molecule library for compounds that block HGF-induced epithelial scattering. Most hits identified in this screen exhibit anti-mitotic properties. Here we assess the biological mechanism of a compound that blocks HGF-induced scattering with limited anti-mitotic activity. Analogs of this compound have one of two distinct activities: inhibiting either cell migration or cell proliferation with cell cycle arrest in G2/M. Each activity bears unique structure–activity relationships. The mechanism of action of anti-mitotic compounds is by inhibition of microtubule polymerization; these compounds entropically and enthalpically bind tubulin in the colchicine binding site, generating a conformational change in the tubulin dimer.     

Regulation of cancer metastasis by microRNAs

MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs that have been found highly conserved among species. MiRNAs are able to negatively regulate gene expression through base pairing of 3’ UTRs of their target genes. Therefore, miRNAs have been shown to play an important role in regulating various cellular activities. Over the past decade, substantial evidences have been obtained to show that miRNAs are aberrantly expressed in human malignancies and could act as “OncomiRs” or “Tumor suppressor miRs”. In recent years, increasing number of studies have demonstrated the involvement of miRNAs in cancer metastasis. Many studies have shown that microRNAs could directly target genes playing a central role in epithelia-mesenchymal-transition (EMT), a cellular transformation process that allows cancer cells to acquire motility and invasiveness. EMT is considered an essential step driving the early phase of cancer metastasis. This review will summarize the recent findings and characterization of miRNAs that are involved in the regulation of EMT, migration, invasion and metastasis of cancer cells. Lastly, we will discuss potential use of miRNAs as diagnostic and prognostic biomarkers as well as therapeutic targets for cancer.     

Sphingosine kinase activation regulates hepatocyte growth factor induced migration of endothelial cells

Hepatocyte growth factor (HGF)-induced migration of endothelial cells is critical for angiogenesis. Sphingosine kinase (SPK) is a key enzyme catalyzing the formation of sphingosine-1-phosphate (S1P), a lipid messenger that is implicated in the regulation of a wide variety of important cellular events through both intracellular and extracellular mechanisms. The aim of this study was to investigate whether activation of SPK is involved in the migration of endothelial cells induced by HGF. The biological functions of HGF are mediated through the activation of its high-affinity tyrosine kinase receptor, c-met protooncogene. In the present study, Treatment of ECV304 endothelial cells with HGF resulted in tyrosine phosphorylation of c-Met and activation of SPK in a concentration-dependent manner. Either Ly294002 or PD98059, specific inhibitor of the PI3K and ERK/MAPK pathways, respectively, blocked the HGF-induced activation of SPK. HGF stimulation significantly increased intracellular S1P level, but no detectable secretion of S1P into the cell culture medium was observed. Treatment of ECV304 cells with pertussis toxin (PTX) has no effect on the HGF-induced migration, indicating extracellular S1P is dispensable for this process. Overexpression of wild-type SPK gene in ECV 304 cells increased the intracellular S1P and enhanced the HGF-induced migration, whereas inhibition of cellular SPK activity by N,N-dimethylsphingosine (DMS), a potent inhibitor of SPK, or by expression of a dominant-negative SPK (DN-SK) blocked the HGF-induced migration of ECV 304 cells. It is suggested that PI3K and ERK/MAPK mediated the activation of SPK and would be involved in the HGF-induced migration of endothelial cells. These results elucidate a novel mechanism by which intracellularly generated S1P mediates signaling from HGF/c-Met to the endothelial cell migration.     

Osteopontin contributes to hepatocyte growth factor-induced tumor growth and metastasis formation

The cytokine hepatocyte growth factor (HGF)/scatter factor-1 and its cognate receptor, Met, are involved in the etiology and progression of many types of cancer. Despite recent advances in understanding the signal transduction pathways activated by HGF, the mechanism by which HGF exerts its tumorigenic effect is not well understood. To identify proteins that may be involved in mediating HGF-induced cell motility, invasiveness, and tumorigenesis, we used two separate differential display screening methods to identify changes in gene expression that are initiated by HGF in an epithelial cell culture system. Among several known and unknown genes whose expression was modified, osteopontin (OPN), a protein previously associated with tumorigenesis, was found to be upregulated within 6 h following HGF stimulation. OPN expression was dependent on activation of the PI-3 kinase pathway. Autocrine secretion of HGF resulted in sustained expression of OPN. Downregulation of opn expression by stable antisense transfection attenuated OPN expression and repressed HGF-induced invasiveness in vitro and decreased HGF-mediated tumor growth and metastasis formation in vivo. Constitutive expression of OPN in itself exerted partial invasiveness in vitro, but its expression itself was not sufficient to initiate tumor growth or metastasis formation in vivo. Thus, together with other molecules, OPN activity contributes to HGF-induced tumor growth and invasiveness.     

Potentiation of invasive activity of hepatoma cells by reactive oxygen species is mediated by autocrine/paracrine loop of hepatocyte growth factor

We have already reported that reactive oxygen species (ROS) promote rat ascites hepatoma cell invasion beneath mesentery-derived mesothelial cell monolayer. To investigate the mechanism for this, we examined the involvement of motility factors, particularly hepatocyte growth factor (HGF). Rat ascites hepatoma cell line of AH109A expressed HGF and c-Met mRNAs. Treatment with ROS augmented amounts of HGF mRNA in AH109A and HGF concentration in the medium. ROS also induced HGF gene expression in mesothelial cells. Exogenously added HGF enhanced invasive activity of AH109A cells, but exerted no effect on proliferation. AH109A cells pretreated with ROS showed an increased invasive activity, which was cancelled by simultaneous pretreatment with anti-HGF antibody. These results suggest that the invasive activity of AH109A is mediated by the autocrine and paracrine pathways of HGF, and ROS potentiate invasive activity by inducing gene expression of HGF in AH109A and mesothelial cells.     

Progressive changes in Met-dependent signaling in a human ovarian surface epithelial model of malignant transformation

We used an experimental in vitro model of human ovarian surface epithelium (OSE), the tissue of origin of >90% of ovarian cancers, to more precisely define the contribution of hepatocyte growth factor (HGF) to various OSE phenotypes at different stages of neoplastic progression. Neoplastic transformation of OSE in cultures was achieved by multiple genetic manipulations, resulting in the nontumorigenic line IOSE-29, the tumorigenic IOSE-Ov29, and the tumor-derived, more highly malignant IOSE-Ov29/T4. We demonstrate here that, compared to IOSE-29, IOSE-Ov29 and IOSE-Ov29/T4 exhibited higher levels of the HGF receptor Met and an increasing duration of ERK1/2 activation with malignant progression, in conjunction with other neoplastic properties. HGF activated Met signaling in all lines but elicited different responses: HGF induced cell dispersion (scattering) and collagen gel invasion in IOSE-Ov29 and IOSE-Ov29/T4 but did not alter the growth pattern of IOSE-29. Inhibition with PD98059 and LY294002 independently prevented HGF-induced invasive growth. Furthermore, our results show that HGF-induced invasion can be mediated through a rapamycin-sensitive p70 S6K cascade, which demonstrates that p70S6K can regulate cell motility in addition to its well-established role in protein synthesis. Taken together, our data correlate specific responses to HGF-mediated signaling with specific signaling pathways and with progressive neoplastic changes.     

Numb is a negative regulator of HGF dependent cell scattering and Rac1 activation

Numb is an endocytic adaptor protein that regulates internalization and post-endocytic trafficking of cell surface proteins. In polarized epithelial cells Numb is localized to the basolateral membrane, and recent work has implicated Numb in regulation of cell adhesion and migration, suggesting a role for Numb in epithelial–mesenchymal transition (EMT). We depleted MDCK cells of Numb and examined the effects downstream of EMT-promoting stimuli. While knockdown of Numb did not affect apicobasal polarity, we show that depletion of Numb destabilizes E-cadherin-based cell–cell adhesion and promotes loss of epithelial cell morphology. In addition, Numb knockdown in MDCK cells potentiates HGF-induced lamellipodia formation and cell dispersal. Examination of Rac1-GTP levels in Numb knockdown cells revealed hyperactivation of Rac1 following extracellular calcium depletion and HGF stimulation, which corresponds with enhanced loss of cell adhesions and lamellipodia formation. Furthermore, inhibition of Rac1 in Numb depleted cells stabilized cell–cell contacts following depletion of extracellular calcium. Together, these data indicate that Numb acts to suppress Rac1-GTP accumulation, and its loss leads to increased sensitivity toward extracellular signals that disrupt cell–cell adhesion to induce epithelial–mesenchymal transition (EMT) and cell dispersal.     

Intracellular signaling cascades triggered by the NK1 fragment of hepatocyte growth factor in human prostate epithelial cell line PNT1A

Hepatocyte Growth Factor (HGF)/c-MET signaling has an emerging role in promoting cell proliferation, survival, migration, wound repair and branching in a variety of cell types. HGF plays a crucial role as a mediator of stromal–epithelial interactions in the normal prostate but the precise biological function of HGF/c-Met interaction in the normal prostate and in prostate cancer is not clear. HGF has two naturally occurring splice variants and NK1, the smallest of these HGF variants, consists of the HGF amino terminus through the first kringle domain. We evaluated the intracellular signaling cascades and the morphological changes triggered by NK1 in human prostate epithelial cell line PNT1A which shows molecular and biochemical properties close to the normal prostate epithelium. We demonstrated that these cells express a functional c-MET, and cell exposure to NK1 induces the phosphorylation of tyrosines 1313/1349/1356 residues of c-MET which provide docking sites for signaling molecules. We observed an increased phosphorylation of ERK1/2, Akt, c-Src, p125FAK, SMAD2/3, and STAT3, down-regulation of the expression of epithelial cell–cell adhesion marker E-cadherin, and enhanced expression levels of mesenchymal markers vimentin, fibronectin, vinculin, α-actinin, and α-smooth muscle actin. This results in cell proliferation, in the appearance of a mesenchymal phenotype, in morphological changes resembling cell scattering and in wound healing. Our findings highlight the function of NK1 in non-tumorigenic human prostatic epithelial cells and provide a picture of the signaling pathways triggered by NK1 in a unique cell line.     

Knockdown of kinesin KIF11 abrogates directed migration in response to epidermal growth factor-mediated chemotaxis

Establishment of microtubule polarity is critical for directional cell migration involved in morphogenesis, differentiation, cell division, and metastasis. Current models, involving iterative microtubule capture and inactivation of microtubule depolymerizing mechanisms at the leading edge, cannot account for the biased migration exhibited by cells in culture in the absence of directional cues, suggesting central mechanisms governing microtubule polarity remain unknown. We engineered two human MDA-MB-231/IMP1 breast carcinoma cell lines, denoted kdKIF11-1 and kdKIF11-2, in which the kinesin KIF11 (also known as Eg5) was stably knocked down by two different shRNAs. Western blot analysis showed knockdown by each shRNA decreased KIF11 expression by 58% and 79% for kdKIF11-1 and kdKIF11-2, respectively, whereas Rac1 expression was unaffected. All cell lines retained a well-defined microtubule structure. Compared to cells infected with the control viral vector, both KIF11 knockdown cell lines displayed a 14–45% increase in cell motility in a scratch wound healing assay. In contrast, KIF11 knockdown decreased invasion by 70%, compared to the control, as measured by invasion through Matrigel-coated transwells. To determine whether the reduction in invasion was due to reduced chemotaxis, we substituted collagen for Matrigel in the transwell assay and similarly observed a 44–54% reduction in migration, using EGF as the chemoattractant. However, when including EGF in both the upper and lower chambers of the transwell to stimulate migration but eliminate chemotaxis, transwell migration decreased for the control cell line only, indicating that KIF11 knockdown did not impair migration, but severely impaired chemotaxis. We conclude KIF11 is a key downstream molecule that responds to directional cues in chemotaxis to govern the direction of migration.     

Hepatocyte growth factor triggers signaling cascades mediating vascular smooth muscle cell migration

A key event in neointima formation and atherogenesis is the migration of vascular smooth muscle cells (VSMCs) into the intima. This is controlled by cytokines and extracellular matix (ECM) components within the microenvironment of the diseased vessel wall. At present, these signals have only been partially identified. In this study, we demonstrate that Met, the receptor tyrosine kinase for hepatocyte growth factor (HGF), is expressed on VSMCs isolated from the intima of atherosclerotic plaques of carotid arteries. Stimulation with HGF led to activation of Met as well as to activation of PI3-K, PKB/Akt, MEK, and the MAP kinases Erk1 and -2. Moreover, HGF induced lamellipodia formation, a characteristic feature of motile cells, and promoted VSMC migration across fibronectin-coated filters. The HGF-induced cell migration was mediated by beta1 integrins and required PI3-K activation. Our results suggest a role for the HGF-Met signaling pathway in the pathogenesis of atherosclerosis and restenosis.     

Regulation of migration of primary prostate epithelial cells by secreted factors from prostate stromal cells

Stromal-epithelial interactions, which regulate the migration of prostate epithelial cells, play an important role in prostate development, prostatic hyperplasia, and prostate cancer. The objective of this study was to determine how the prostate stroma stimulates the migration of primary prostate epithelial cells (PECs). In the Boyden chamber assay, PEC migration was strongly induced by the conditioned medium of primary prostate stromal cells (PSC-CM). Stimulation of PEC migration depended on the concerted action of adhesion and motility factors in the PSC-CM. Immobilized proteins from PSC-CM mediated adhesion, spreading, and head-to-tail polarization of PECs. Migration induced by immobilized PSC-CM proteins was significantly increased by hepatocyte growth factor/scatter factor (HGF/SF). Inhibition of P13-kinase or Src-family kinases, but not MEK or PLCchi, abolished migration in the Boyden chamber assay. Consistent with their concerted activity in migration assays, the combination of adhesion and motility factors was required for efficient activation of the P13-kinase/Akt pathway. HGF/SF in the PSC-CM was the principal stimulator of the P13-kinase/Akt pathway and an important mediator of PSC-CM-induced PEC migration. In conclusion, our data show that the migration of primary PECs is regulated by the P13-kinase and Src-family kinase signaling pathways and that the activation of the P13-kinase pathway requires adhesion and motility factors from the prostate stroma.     

Connective tissue growth factor (CTGF) and cancer progression

Connective tissue growth factor (CTGF) is a member of the CCN family of secreted, matrix-associated proteins encoded by immediate early genes that play various roles in angiogenesis and tumor growth. CCN family proteins share uniform modular structure which mediates various cellular functions such as regulation of cell division, chemotaxis, apoptosis, adhesion, motility, angiogenesis, neoplastic transformation, and ion transport. Recently, CTGF expression has been shown to be associated with tumor development and progression. There is growing body of evidence that CTGF may regulate cancer cell migration, invasion, angiogenesis, and anoikis. In this review, we will highlight the influence of CTGF expression on the biological behavior and progression of various cancer cells, as well as its regulation on various types of protein signals and their mechanisms.     

Ixodid tick salivary gland products target host wound healing growth factors

For successful blood-feeding, ticks must confront the host immune system comprising many cells and signaling molecules, mainly cytokines and growth factors. These factors bind to specific receptors on the cell membranes, thereby initiating a signaling cascade that leads to distinct cellular activities. Ticks are able to manipulate host immune responses via molecules secreted from their salivary glands. Saliva of ixodid ticks contains factors binding important cytokines and their subgroup, chemokines. Here we demonstrate that constituents of tick salivary gland extract (SGE) also appear to bind growth factors: transforming growth factor beta (TGF-β1), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF-2), and hepatocyte growth factor (HGF), depending on tick species. SGE derived from Amblyommavariegatum reacted with TGF-β1, PDGF, FGF-2 and HGF; Dermacentorreticulatus and Rhipicephalusappendiculatus with TGF-β1, FGF-2 and HGF; and Ixodes ricinus and Ixodesscapularis with PDGF. SGE from the species targeting PDGF (A. variegatum and I. ricinus) also inhibited cell proliferation in vitro and induced a change in morphology of different cell lines. These effects correlated with disruption of the actin cytoskeleton. Such effects were not observed with SGE of the two species that did not target PDGF. Targeting of wound healing growth factors appears to be yet another strategy ixodid ticks adopt for suppression of inflammation and successful haematophagy.     

Mechanisms and significance of bifunctional NK4 in cancer treatment

Based on the background that hepatocyte growth factor (HGF) and c-Met/HGF receptor tyrosine kinase play a definite role in tumor invasion and metastasis, NK4, four-kringles containing intramolecular fragment of HGF, was isolated as a competitive antagonist for the HGF-c-Met system. Independent of its HGF-antagonist action, NK4 inhibited angiogenesis induced by vascular endothelial cell growth factor and basic fibroblast growth factor, as well as HGF, indicating that NK4 is a bifunctional molecule that acts as an HGF-antagonist and angiogenesis inhibitor. Interestingly, kringle domains in distinct types of proteins, e.g., plasminogen, prothrombin, plasminogen activators, apolipoprotein(a), and HGF, share angioinhibitory actions. In experimental models of distinct types of cancers, NK4 protein administration or NK4 gene therapy inhibited tumor invasion, metastasis, and angiogenesis-dependent tumor growth. Cancer treatment with NK4 may prove to suppress malignant tumors to be 'static' in both tumor growth and spreading, as based on biological characteristics of malignant tumors.     

Nischarin inhibits Rac induced migration and invasion of epithelial cells by affecting signaling cascades involving PAK

Nischarin, a cytosolic protein that binds the alpha5beta1 integrin, plays an important role in fibroblast migration, and in regulation of the actin cytoskeleton. The effect of Nischarin on Rac induced migration and invasion by breast and colon epithelial cell lines has been determined. In these cells, Rac potently induced migration, as well as invasion of matrix; both of these events were strongly inhibited by overexpression of Nischarin. To understand the mechanism of Nischarin's inhibitory role in Rac induced cell migration, several effector domain mutants of Rac1 were employed. Nischarin was able to inhibit migration induced by Rac effector mutants that can activate PAK and JNK, but not migration stimulated by other Rac mutants. Further, Nischarin inhibited PAK induced cell migration, while not affecting migration induced by MEKK1, a Rac effector in the JNK pathway. In addition, Nischarin failed to inhibit migration induced by MEK1, a downstream effector in the Ras-Raf-MEK-Erk signaling cascade. Furthermore, Nischarin does not affect Rac mediated JNK and PI3K activities. However, Rac induced migration and invasion were effectively blocked by pharmacological inhibitors of PI-3 kinase and MEK. These results suggest that several pathways contribute to cell migration, but that Nischarin selectively inhibits Rac driven signaling cascades that affect migration through PAK.     

Promoter-independent regulation of vimentin expression in mammary epithelial cells by val(12)ras and TGFbeta

The 1,029 series of mammary epithelial cell lines (D6, GP+E, r3 and r3T) are progressively more transformed: the latter two by val(12)ras. These cell lines respond to TGFbeta by undergoing early events of epithelial-mesenchymal transition (EMT), including morphological changes and redistribution of E-cadherin. Tumors formed by r3T cells in the choroid of the eye express vimentin, a late marker of EMT, possibly in response to TGFbeta. In vitro, vimentin expression is induced in all the cell lines by TGFbeta treatment, whereas cytokeratin expression is only slightly affected. Surprisingly, ras transformation results in a 10-fold suppression of vimentin expression. Neither suppression of vimentin by ras transformation nor induction by TGFbeta is mediated by the vimentin promoter in r3T cells. In transient transfection assays, several human vimentin promoter constructs are more active in the low-expressing r3T cell line than in the vimentin-expressing mesenchymal cell line NIH3T3. In the r3T cells, there is no effect of TGFbeta treatment for 9 days on the activity of either promoter. Azacytidine treatment does not affect vimentin expression in either NIH3T3 or r3T, suggesting that promoter methylation is not the mechanism of suppression by ras. Finally, the half-life of the vimentin mRNA is similar in both the r3T cells and NIH3T3 cells. We conclude that the suppression of vimentin expression by ras, and the relief of this suppression by TGFbeta, occurs in a promoter-independent fashion, possibly through sequences in the first or second intron.     

Bone morphogenetic protein-4 abrogates lumen formation by mammary epithelial cells and promotes invasive growth

Bone morphogenetic proteins (BMPs) are multifunctional cytokines that regulate key developmental processes, but are also overexpressed in many carcinomas. To assess whether BMPs would influence the three-dimensional architecture of epithelial structures, we took advantage of an in vitro model in which mammary epithelial cells form alveolar-like spherical cysts in collagen gels. We found that BMP-4 has a dramatic, biphasic effect on the organization of epithelial cysts. When added in the concentration range of 1-10 ng/ml, the cytokine abrogates lumen formation and induces the outgrowth of multiple invasive cord-like structures. At higher concentrations (20-100 ng/ml), BMP-4 additionally disrupts cell-cell adhesion, resulting in cyst disintegration and scattering of individual cells into the surrounding collagen matrix. The finding that BMP-4 subverts the ability of mammary epithelial cells to form polarized lumen-containing structures and endows them with invasive properties supports the involvement of this cytokine in the progression of breast cancer.