Platelet-derived Growth Factor Receptor-β Research Articles

A Novel Signaling Axis of Matriptase/PDGF-D/β-PDGFR in Human Prostate Cancer

Increasing evidence indicates the significance of platelet-derived growth factor receptor-β (β-PDGFR) signaling in prostate cancer (PCa). Accordingly, preclinical studies suggest the potential of β-PDGFR as a therapeutic target in metastatic PCa. However, a ligand responsible for β-PDGFR activation in PCa was unknown, and recent clinical trials with imatinib mesylate showed limited success due to normal tissue toxicity. Similarly, in spite of mounting evidence indicating the significance of matriptase in PCa, little is known about its substrates or molecular actions during PCa progression. Here, we identified PDGF-D as a ligand for β-PDGFR in PCa and discovered matriptase as its regulator. Matriptase activates PDGF-D by proteolytic removal of the CUB domain in a 2-step process, creating a hemidimer, followed by growth factor domain dimer (GFD-D) generation. Matriptase can deactivate PDGF-D by further proteolytic cleavage within the GFD, revealing its biphasic regulation. Importantly, PDGF-D/matriptase colocalization is accompanied with β-PDGFR phosphorylation in human PCa tissues. This study unveiled a novel signaling axis of matriptase/PDGF-D/β-PDGFR in PCa, providing new insights into functional interplay between serine protease and growth factor signaling networks.

Anthocyanins isolated from the purple-fleshed sweet potato attenuate the proliferation of hepatic stellate cells by blocking the PDGF receptor

During the process of liver fibrosis, hepatic stellate cells (HSCs) play a critical role in the increased formation and reduced degradation of extracellular matrix in the liver. We investigated the anti-proliferative effects of an anthocyanin fraction (AF), isolated from the purple-fleshed sweet potato, on platelet-derived growth factor (PDGF)-BB-dependent signaling pathways in HSC-T6 cells. HSC proliferation plays a pivotal role in liver fibrogenesis. The AF suppressed HSC activation, including PDGF-induced proliferation and α-smooth muscle actin (α-SMA) expression. Additionally, AF inhibited PDGF-BB-induced Akt and ERK1/2 phosphorylation. AF inhibited the phosphorylation level of PDGF receptor-β (PDGFR-β) following PDGF-BB stimulation, providing a mechanism for the inhibition of AF-mediated kinase. These results suggest that AF suppresses HSC proliferation by blocking PDGFR-β signaling, inhibiting Akt and ERK1/2 activation and α-SMA expression.

Establishment, Characterization, and Drug Sensitivity of a New Ewing Sarcoma Cell Line (SS-ES-1)

Ewing sarcoma (ES) is one of the most malignant tumors in children and young adults. We present here a new ES cell line, SS-ES-1, established from the left thoracic tumor of a 16-year-old female patient. The SS-ES-1 cells retained genotype, morphology, and growth rate for over 150 passages. Immunocytochemical staining showed the strong immunoreactivity for cytokeratin, epithelial membrane antigen, neurofilament, CD99, P53, Ki-67, platelet-derived growth factor receptor-β, estrogen receptor-α (ER-α), and Bcl-2, but no reactivity for glial fibrillary acidic protein, epidermal growth factor receptor, and HER-2/neu. The presence of the type 1 EWS/FLI-1 fusion transcripts was confirmed by reverse transcriptase-polymerase chain reaction. On the basis of the MTT assay results, GW2974, a dual inhibitor of epidermal growth factor receptor and HER-2/neu, exhibited only a weak cytotoxic response in SS-ES-1 cells. In contrast, tyrphostin A9, a specific inhibitor of platelet-derived growth factor receptor, had a high cytotoxic effect against these cells. Surprisingly, it was found that SS-ES-1 cells displayed a high sensitivity to 4OH-tamoxifen. In conclusion, the SS-ES-1 cell line shows unique cellular properties, which makes it a useful model for studying various aspects of the biology of ES. In addition, the results suggest that ER can be a good therapeutic target for ER + ES.

Aspirin-Triggered Lipoxin and Resolvin E1 Modulate Vascular Smooth Muscle Phenotype and Correlate with Peripheral Atherosclerosis

Atherosclerosis is a chronic inflammatory disease of the vessel wall. Recent evidence suggests that chronic vascular inflammation ensues as an imbalance between pro- and anti-inflammatory mediators. Recently identified lipid mediators (eg, lipoxins and resolvins) play active roles in promoting the resolution of inflammation. Alterations in vascular smooth muscle cell (VSMC) phenotype, which manifest as a loss of contractile protein expression and increased proliferation and migration, are prominent mechanistic features of both atherosclerosis and restenosis following various interventions (eg, angioplasty and bypass grafting). We sought to determine whether human atherosclerosis is associated with a "resolution deficit" and whether lipoxins and resolvins influence VSMC phenotype. Here we report that plasma levels of aspirin-triggered lipoxin are significantly lower in patients with symptomatic peripheral artery disease than in healthy volunteers. Both aspirin-triggered lipoxin and resolvin E1 block platelet-derived growth factor-stimulated migration of human saphenous vein SMCs and decrease phosphorylation of the platelet-derived growth factor receptor-β. Importantly, receptors for aspirin-triggered lipoxin and resolvin E1 (ALX and ChemR23, respectively) were identified in human VSMCs. Overall, these results demonstrate that stimulatory lipid mediators confer a protective phenotypic switch in VSMCs and elucidate new functions for these mediators in the regulation of SMC biology. These results also suggest that peripheral artery disease is associated with an inflammation-resolution deficit and highlight a potential therapeutic opportunity for the regulation of vascular injury responses.

Fibrogenic cell phenotype modifications during remodelling of normal and pathological human liver in cultured slices

The debate concerning the potential remodelling and/or reversibility of cirrhotic lesions and biliary fibrosis is still open. In this work, we have used the precision-cut liver slice (PCLS) model, which maintains cell-cell and cell-matrix interactions to study, by immunohistochemistry, the behaviour of the different fibrogenic cells, i.e. hepatic stellate cells (HSC) and portal fibroblasts, in cultured (for 1 week) PCLS derived from normal and fibrotic human livers. In normal liver, before and after culture, α-smooth muscle (SM) actin was present only in the vessel walls. Platelet-derived growth factor (PDGF) receptor-β was expressed before and after culture by portal fibroblasts, and appeared after culture in HSC. Before culture, CD 34 was not expressed in parenchyma, but appeared after culture in sinusoidal endothelial cells. In cirrhotic lesions, before culture, α-SM actin, PDGF receptor-β and Thy-1 were expressed in septa; after culture, α-SM actin expression disappeared but the expression of the PDGF receptor-β and Thy-1 was maintained. In cholestatic liver specimens, α-SM actin, PDGF receptor-β and Thy-1 expression, which was present before culture in enlarged portal areas, disappeared after culture, and apoptosis was detected. In the parenchyma of both cirrhotic and cholestatic livers, the expression of the PDGF receptor-β and of CD 34, which was not observed before culture, was present in HSC and sinusoidal endothelial cells, respectively, after culture. These results indicate that during remodelling of pathological tissues in cultured liver slices, the myofibroblastic cells derived from HSC or from portal fibroblasts show different behaviours, suggesting different mechanisms of activation/deactivation.

12/15-lipoxygenase–derived lipid peroxides control receptor tyrosine kinase signaling through oxidation of protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs) are regulated through reversible oxidation of the active-site cysteine. Previous studies have implied soluble reactive oxygen species (ROS), like H(2)O(2), as the mediators of PTP oxidation. The potential role(s) of peroxidized lipids in PTP oxidation have not been described. This study demonstrates that increases in cellular lipid peroxides, induced by disruption of glutathione peroxidase 4, induce cellular PTP oxidation and reduce the activity of PDGF receptor targeting PTPs. These effects were accompanied by site-selective increased PDGF beta-receptor phosphorylation, sensitive to 12/15-lipoxygenase (12/15-LOX) inhibitors, and increased PDGF-induced cytoskeletal rearrangements. Importantly, the 12/15-LOX-derived 15-OOH-eicosatetraenoic acid lipid peroxide was much more effective than H(2)O(2) in induction of in vitro PTP oxidation. Our study thus establishes that lipid peroxides are previously unrecognized inducers of oxidation of PTPs. This identifies a pathway for control of receptor tyrosine kinase signaling, which might also be involved in the etiology of diseases associated with increased lipid peroxidation.

Protein tyrosine phosphatase PTPεM negatively regulates PDGF β-receptor signaling induced by high glucose and PDGF in vascular smooth muscle cells

Vascular smooth muscle cell (VSMC) proliferation and migration and vascular endothelial cell (VEC) dysfunction are closely associated with the development of atherosclerosis. We previously demonstrated that protein tyrosine phosphatase ε M (PTPεM) promotes VEC survival and migration. The present study investigates the biological functions of PTPεM in VSMCs and determines whether PTPεM is implicated in diabetes-accelerated atherosclerosis. We overexpressed wild-type and inactive PTPεM and an small interfering RNA (siRNA) of PTPεM by using an adenovirus vector to investigate the effects of PTPεM upon platelet-derived growth factor (PDGF)- and high glucose (HG)-induced responses of rat VSMCs in vitro. We found that PTPεM decreased PDGF-induced DNA synthesis and migration by reducing the phosphorylation level of the PDGF β-receptor (PDGFRβ) with subsequently suppressed H(2)O(2) generation. The HG content in the medium generated H(2)O(2), upregulated PDGFRβ expression and its tyrosine-phosphorylation, and elevated NADPH oxidase 1 (Nox1) expression even without exogenous PDGF, all of which were downregulated by PTPεM. The PDGFR inhibitor AG1296 also blocked HG-induced Nox1 expression and H(2)O(2) production. Moreover, HG suppressed PTPεM expression itself, which was blocked by the antioxidant N-acetyl-l-cysteine. The effects of PTPεM siRNA were the opposite of those of wild-type PTPεM. Therefore, PTPεM negatively regulates PDGFRβ-mediated signaling pathways that are crucial for the pathogenesis of atherosclerosis, and PTPεM may be involved in diabetes-accelerated atherosclerosis.

Mitogenic factors promoting intestinal smooth muscle cell proliferation

Intestinal smooth muscle cells are normally quiescent, but in the widely studied model of trinitrobenzene sulfonic acid (TNBS)-induced colitis in the rat, the onset of inflammation causes proliferation that leads to increased cell number and an altered phenotype. The factors that drive this are unclear and were studied in primary cultures of circular smooth muscle cells (CSMC) from the rat colon. While platelet-derived growth factor (PDGF)-AA, fibroblast growth factor (FGF), and epidermal growth factor (EGF) were ineffective, PDGF-BB and insulin-like growth factor-1 (IGF-1) caused significant increase in [(3)H]thymidine incorporation, bromodeoxyuridine uptake, and increased CSMC number, with PDGF-BB (≥0.2 nM) substantially more effective than IGF-1. Surprisingly, CSMC lacked expression of PDGF receptor-β (PDGF-Rβ) upon isolation but by 4 days in vitro, CSMC gained expression of PDGF-Rβ as shown by quantitative PCR, Western blot analysis, and immunocytochemistry; these CSMC responded to PDGF-BB but not IGF-1. PDGF-BB caused PDGF-Rβ phosphorylation and mobilization from the surface membrane, leading to activation of both Akt and ERK signaling pathways, which were essential for subsequent proliferation. In contrast, PDGF-AA, FGF, EGF, and IGF-1 were ineffective. In vivo, control CSMC lacked expression of PDGF-Rβ. However, this changed rapidly with TNBS-colitis, and by day 2 when CSMC proliferation in vivo is maximal, freshly isolated CSMC showed on-going PDGF-Rβ phosphorylation that was further increased by exogenous PDGF-BB. This suggests that the onset of PDGF-Rβ expression is a key factor in CSMC growth in vitro and in vivo, where inflammation may damage intrinsic inhibitory mechanisms and thus lead to hyperplasia.

PDGF β-Receptor Kinase Activity and ERK1/2 Mediate Glycosaminoglycan Elongation on Biglycan and Increases Binding to LDL

The initiation of atherosclerosis involves the subendothelial retention of lipoproteins by proteoglycans (PGs). Structural characteristics of glycosaminoglycan (GAG) chains on PGs influence lipoprotein binding and are altered adversely by platelet-derived growth factor (PDGF). The signaling pathway for PDGF-mediated GAG elongation via the PDGF receptor (PDGFR) was investigated. In human vascular smooth muscle cells, PDGF significantly increased (35)S-sulfate incorporation into PGs and GAG chain size. PGs from PDGF-stimulated cells showed increased binding low-density lipoprotein (P < 0.001) in gel mobility shift assays. Knockdown of PDGFRbeta using small interfering RNA demonstrated that PDGF mediated changes in PGs via PDGFRbeta. GAG synthesis and hyperelongation was blocked by inhibition of receptor tyrosine kinase autophosphorylation site Tyr857 activity using Ki11502 or imatinib. Downstream signaling to GAG hyperelongation was mediated through ERK MAPK and not phosphatidylinositol-3 kinase or phospholipase Cgamma. In high-fat-fed apolipoprotein E(-/-) mice, inhibition of PDGFRbeta activity by imatinib reduced aortic total lipid staining area by 35% (P < 0.05). Inhibition of PDGFRbeta tyrosine kinase activity leads to inhibition of GAG synthesis on vascular PGs and aortic lipid area in vivo. PDGFRbeta and its signaling pathways are potential targets for novel therapeutic agents to prevent the earliest stages atherosclerosis.

Elevated expression of c-kit in small venous malformations of blue rubber bleb nevus syndrome

The blue rubber bleb nevus syndrome (BRBNS, syn. bean syndrome) is a rare disease characterized by multiple cutaneous and gastrointestinal venous malformations associated with severe bleeding. However, the underlying molecular mechanisms are unknown and no targeted therapeutic approach exists to date. Here we report the case of a 19-year-old male patient with severe BRBNS in whom we analyzed the expression of tyrosine kinases frequently involved in tumor development by immunohistochemistry (vascular endothelial growth factor receptor-2, stem cell growth factor receptor (c-kit), platelet-derived growth factor receptor-β, and stem cell tyrosine kinase-1). A prominent expression of c-kit was detectable in smaller blood vessels, which also showed a moderate expression of the proliferation marker MIB1. Surprisingly, other growth factor receptors stained negatively. We therefore conclude that pharmacological inhibition of the c-kit signaling pathway in cavernous hemangiomas by selective kinase inhibitors may offer options in the treatment of BRBNS patients.

The transcriptional repression of platelet-derived growth factor receptor-β by the zinc finger transcription factor ZNF24

The zinc finger transcription factor ZNF24 is a relatively uncharacterized Krüppel-like transcription factor, displaying a suppressive effect on the transcription. ZNF24 is involved in negative regulation of vascular endothelial growth factor (VEGF) and may represent a novel repressor of VEGF transcription. In a previous study, our analysis reveals that ZNF24 is a pleiotropic factor that has a role in hematopoiesis, brain development and cancers. Activation of PDGFR-β is critically involved into various cell functions including recruitment of stromal cells and VEGF induction in tumor and perivascular cells. Here, overexpression of ZNF24 resulted in a significant down-regulation of PDGFR-β, whereas silencing of ZNF24 with small interfering RNA led to increased PDGFR-β expression. Overexpression of ZNF24 resulted in a significant decrease in PDGFR-β promoter activity. These data suggest that ZNF24 is involved in negative regulation of PDGFR-β and may represent a novel repressor of PDGFR-β transcription.

Phase I study of dasatinib in combination with bevacizumab in advanced solid tumors.

TPS163 Background: Despite the tremendous promise of new signal transduction and angiogenesis inhibitors, these targeted drugs have met with mixed results so far in the clinic. In solid tumors, inhibition of multiple targets may be necessary for significant clinical effects. Dasatinib is an inhibitor of all SRC-family kinases, as well as BCR-ABL, c-KIT, multiple EPH-family kinases, PDGFβ receptor and other tyrosine kinases. Bevacizumab is a humanized IgG1 monoclonal antibody that binds all biologically active isoforms of human vascular endothelial growth factor (VEGF) with high affinity. Attenuation of both c-Src and VEGF pathways simultaneously provides the potential for synergistic antitumor activity, as there is a extensive interaction between these pathways. VEGFR transmits its downstream signal through multiple pathways involving c-Src (including activation of the prosurvival FAK pathway). Alternatively, VEGF production is known to be Src-mediated (including via STAT5 signaling). Thus, inhibition of ...

Mutation of tyrosine residue 857 in the PDGF β-receptor affects cell proliferation but not migration

Activation of platelet-derived growth factor (PDGF) receptors occurs through ligand-induced dimerization and autophosphorylation. In this study, we investigated the effects of mutation of tyrosine residue 857 (Y857) in the activation loop of the PDGF β-receptor (PDGFRβ) to phenylalanine (Y857F). In agreement with previous observations, we found that PDGFRβ Y857F had a severely diminished in vitro kinase activity. However, in vivo the overall amount of tyrosine phosphorylation of PDGFRβ Y857F was similar to that of the wild-type receptor, except for the tyrosine residue 771 (Y771) which displayed a stronger phosphorylation in the mutant receptor. Analysis of the ability to induce signal transduction revealed that the PDGFRβ Y857F mutant had an attenuated activation of Akt and Erk1/2 MAP kinase. In contrast, the mutant receptor efficiently mediated phosphorylation of the ubiquitin-ligase c-Cbl that participates in receptor internalization and degradation, and PLCγ which has previously been shown to be connected with various cellular responses, including migration. However, the protein tyrosine phosphatase SHP-2, implicated in the PDGF-induced mitogenic response, together with the adaptor proteins Alix and Stam, involved in intracellular sorting of receptor, was not phosphorylated in cells expressing PDGFRβ Y857F. We found that both receptor variants were internalized from the cell surface and degraded at a comparable rate. Interestingly, PDGFRβ Y857F was unable to mediate PDGF-BB-induced mitogenic signaling, whereas it could elicit a chemotactic response.

Mesenchymal stem cells enhance growth and metastasis of colon cancer

Recently, mesenchymal stem cells (MSCs) were reported to migrate to tumor stroma as well as injured tissue. We examined the role of human MSCs in tumor stroma using an orthotopic nude mice model of KM12SM colon cancer. In in vivo experiments, systemically injected MSCs migrated to the stroma of orthotopic colon tumors and metastatic liver tumors. Orthotopic transplantation of KM12SM cells mixed with MSCs resulted in greater tumor weight than did transplantation of KM12SM cells alone. The survival rate was significantly lower in the mixed-cell group, and liver metastasis was seen only in this group. Moreover, tumors resulting from transplantation of mixed cells had a significantly higher proliferating cell nuclear antigen labeling index, significantly greater microvessel area and significantly lower apoptotic index. Splenic injection of KM12SM cells mixed with MSCs, in comparison to splenic injection of KM12SM cells alone, resulted in a significantly greater number of liver metastases. MSCs incorporated into the stroma of primary and metastatic tumors expressed α-smooth muscle actin and platelet-derived growth factor receptor-β as carcinoma-associated fibroblast (CAF) markers. In in vitro experiments, KM12SM cells recruited MSCs, and MSCs stimulated migration and invasion of tumor cells through the release of soluble factors. Collectively, MSCs migrate and differentiate into CAFs in tumor stroma, and they promote growth and metastasis of colon cancer by enhancing angiogenesis, migration and invasion and by inhibiting apoptosis of tumor cells.

The effect of prenatal nicotine exposure on PDGFR-mediated anti-apoptotic mechanism in the caudal brainstem of developing rat

Maternal cigarette smoking is a major risk factor for sudden infant death syndrome (SIDS); however, the mechanism underlying this association is currently unknown. Prenatal nicotine exposure is accompanied by a decrease in the magnitude of hypoxic ventilatory depression, the component of hypoxic ventilatory response that activates the PDGF-β receptor (PDGFR) and its downstream anti-apoptotic cascade in the caudal brainstem (CB) of developing rats. In this study, we evaluated the effect of prenatal nicotine exposure on PDGFR activation and the subsequent activation of downstream anti-apoptotic processes through the Akt/BAD pathway. The 5-day timed-pregnant Sprague–Dawley rats underwent surgical implantation of an osmotic pump containing either normal saline (control) or a solution of nicotine tartrate. The CB was harvested from 5-day-old rat pups ( n = 8–10 for each time point) in each group after exposure to normoxia or hypoxic challenges with 10% O 2 for 5, 15, 30, 60 or 120 min. Immunoprecipitation and immunoblots of CB lysates revealed phosphorylation of PDGFR, Akt and BAD-136 during hypoxia in control pups. Prenatal nicotine exposure was associated with attenuation of these responses at all time points. Analysis of an early apoptotic marker in the CB revealed that activation of cleaved caspase-3 occurred only at 120 min of hypoxic exposure in the control. Prenatal nicotine exposure accelerated this response, causing early activation at 30 and 60 min. We conclude that prenatal nicotine exposure attenuates the phosphorylation of PDGFR, Akt and Bad-136 during hypoxia in the CB of developing rats. This modulation of anti-apoptotic cascades accelerates activation of the early apoptotic marker. We speculate that prenatal nicotine exposure affects apoptosis in the CB of developing animals and may increase the vulnerability of neural cells in the respiratory control area, a process that may underlie the association between maternal smoking and SIDS.

Low Density Lipoprotein Receptor-related Protein 1 (LRP1) Forms a Signaling Complex with Platelet-derived Growth Factor Receptor-β in Endosomes and Regulates Activation of the MAPK Pathway

In addition to its endocytic function, the low density lipoprotein receptor-related protein 1 (LRP1) also contributes to cell signaling events. In the current study, the potential of LRP1 to modulate the platelet-derived growth factor (PDGF) signaling pathway was investigated. PDGF is a key regulator of cell migration and proliferation and mediates the tyrosine phosphorylation of LRP1 within its cytoplasmic domain. In WI-38 fibroblasts, PDGF-mediated LRP1 tyrosine phosphorylation occurred at 37 degrees C but not at 4 degrees C, where endocytosis is minimized. Furthermore, blockade of endocytosis with the dynamin inhibitor, dynasore, also prevented PDGF-mediated LRP1 tyrosine phosphorylation. Immunofluorescence studies revealed co-localization of LRP1 with the PDGF receptor after PDGF treatment within endosomal compartments, whereas surface biotinylation experiments confirmed that phosphorylated LRP1 primarily originates from intracellular compartments. Together, the data reveal the association of these two receptors in endosomal compartments where they form a signaling complex. To study the contribution of LRP1 to PDGF signaling, we used mouse embryonic fibroblasts genetically deficient in LRP1 and identified phenotypic changes in these cell lines in response to PDGF stimulation by performing phospho-site profiling. Of 38 phosphorylated proteins analyzed, 8 were significantly different in LRP1 deficient fibroblasts and were restored when LRP1 was expressed back in these cells. Importantly, the results revealed that LRP1 expression is necessary for PDGF-mediated activation of ERK. Overall, the studies reveal that LRP1 associates with the PDGF receptor in endosomal compartments and modulates its signaling properties affecting the MAPK and Akt/phosphatidylinositol 3-kinase pathways.

Response of malignant scalp dermatofibrosarcoma to presurgical targeted growth factor inhibition

Dermatofibrosarcoma protuberans (DFSP) is an uncommon, locally aggressive, malignant cutaneous tumor that sparingly presents on the scalp. Dermatofibrosarcomas often result from the formation of a fusion oncogene on translocated or supernumerary ring chromosomes 17 and 22, causing the overexpression of PDGFRbeta driven by the COL1A1 promoter. Because of uncertainty surrounding appropriate treatment of aggressive scalp DFSP, the authors performed an extensive review of the available data from a MEDLINE (Ovid) search to describe the clinical presentation and treatment options for this rare tumor. Their search identified 39 different cases, including the illustrative case presented in this study. Adjuvant therapy for this malignant lesion is not universally established in the literature. In the present case, the authors successfully treated a locally invasive scalp DFSP with presurgical therapy that specifically inhibited the PDGFbeta receptor. Imatinib significantly shrank the DFSP tumor mass, reduced hypervascularity, reduced metabolic activity on PET scanning, and permitted a safe gross-total resection. Although wide excision and Mohs micrographic surgery remain the standard surgical treatments for DFSP, the authors illustrate that presurgical chemotherapeutic treatment by imatinib provides a critical adjunct to traditional therapy.

Abstract 4255: Significance of mesenchymal stem cells in growth and metastasis of colon cancer

Abstract Background: Recently, mesenchymal stem cells (MSCs) are reported to migrate to tumor sites as well as sites of injury and to incorporate into tumor stroma, but the effects of interactions between MSCs and tumor cells and the mechanisms underlying these effects remain unclear. We examined the role of MSCs in the tumor micro-environment using an orthotopic colon cancer model and a model of liver metastasis from colon cancer. Materials and Methods: Human MSCs labeled with fluorescent dye were injected into tail veins of nude mice bearing KM12SM human colon cancer cells. KM12SM cells alone or KM12SM cells mixed with MSCs were transplanted into the cecal wall or spleen of nude mice. Differentiation of MSCs in primary colon tumors and metastatic liver tumors were also analyzed. Migration assay and invasion assay were performed to examine attraction between KM12SM cells and MSCs in vitro. Results: In in vivo experiments, systemically injected MSCs migrated to the stroma of orthotopic colon tumors and metastatic liver tumors. Orthotopic transplantation of KM12SM cells mixed with MSCs resulted in significantly greater tumor weight than did transplantation of KM12SM cells alone (p &amp;lt; 0.05). The survival rate was significantly lower in the mixed-cell group, and liver metastasis was seen only in this group (p &amp;lt; 0.001). Moreover, tumors resulting from transplantation of mixed cells had a significantly higher proliferating cell nuclear antigen labeling index (p &amp;lt; 0.01), significantly greater microvessel area (p &amp;lt; 0.01), and significantly lower apoptotic index (p &amp;lt; 0.001). Splenic injection of KM12SM cells mixed with MSCs resulted in a significantly greater number of liver metastases than did splenic injection of KM12SM cells alone (p &amp;lt; 0.01). MSCs incorporated into the stroma of primary and metastatic tumors expressed α-smooth muscle actin and platelet-derived growth factor receptor-β as carcinoma-associated fibroblast (CAF) markers. In in vitro experiments, KM12SM cells recruited MSCs, and MSCs stimulated migration and invasion of tumor cells through the release of soluble factors (p &amp;lt; 0.05, respectively). Conclusion: MSCs migrate and differentiate into CAFs in tumor stroma, and they promote growth and metastasis of colon cancer by enhancing angiogenesis and by inhibiting apoptosis of tumor cells. 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 4255.

Abstract 398: Molecular mechanisms of recurrence of PDGFB-dependent glioma in mice following inhibition of platelet-derived growth factor B expression

Abstract Molecularly targeted therapies hold great promise for the treatment of cancer. Conditional transgenic mouse models offer unique opportunities for investigating whether the targeted inactivation of an oncogene is sufficient for tumor regression and for developing strategies to overcome the mechanisms by which tumors become resistant to targeted therapies. We developed transgenic mice (GFAP/tTA:TRE/PDGFB) in which human platelet-derived growth factor B (hPDGFB) is expressed in a doxycycline responsive manner. Without doxycycline these mice expressed PDGFB in the CNS and developed glioma. Doxycycline administration led to symptomatic recovery in the majority of tumor-bearing mice. However, sustained regression did not occur in 8 of 22 mice following 3 months of doxycycline treatment. In other experiments we implanted spheroid cells derived from other spontaneous mouse glioma arising in this model into the flanks of immuno-deficient mice. We initially observed a significant reduction in the size of established tumors with doxycycline administration, but residual cells from 5 out of 9 tumors began to grow rapidly following a prolonged period of the treatment. In vitro, spheroid cells derived from the relapsed tumors grew in a PDGFB-independent manner. Doxycycline addition to spheroid cultures effectively inhibited hPDGFB expression but did not inhibit proliferation or induce apoptosis. Examination of signaling pathways revealed that PDGFB receptor phopshorylation was significantly reduced by doxycycline but downstream AKT and Erk phosphorylation was sustained. Ongoing experiments are being conducted to identify upstream regulators of these signal transducers. (This work was supported in part by the Theodora B. Betz Foundation and the Andy Fund) 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 398.

957 INHIBITION OF HEPATIC STELLATE CELL ACTIVATION FOLLOWING ANTI-IGFBPRP1 ADMINISTRATION TO THIOACETAMIDE-TREATED MICE

Background and Aim: HSC have been taken centre stage of liver fibrogenesis due to their remarkable synthesis of extracellular matrix proteins and secretion of profibrogenic mediators. MicroRNAs (miRNAs) are small noncoding RNAs, that posttranscriptionally inhibit gene expression by targeting the untranslated region (UTR) of various transcripts. Recently, we have shown that miR-29a and miR-29b reduce collagen expression in HSC. In the present study, we now investigated the role of miR-29 during the expression of profibrogenic growth factors. Methods: miR-29 expression was analysed by Real Time PCR in HSC after stimulation with Hepatocyte Growth Factor (HGF) and in rat liver after four weeks bile-duct obstruction (N=22 BDL and N=13 sham). In order to identify fibrogenic mediators as putative targets of miR-29a and miR-29b, different databases were screened. The putative targets were subsequently analysed by reporter assays. For this purpose the psiCheck vector providing endogenous normalisation of the luciferase reporter activity was used and putative miR-29 binding sites of the identified UTRs were cloned downstream to the luciferase reporter gene. Ago-miR-29, mimicking the miR-29a, were transfected into HSC and synthesis of identified targets were determined. Results: miR-29a and miR-29b were downregulated in rat livers suffering from cholestasis induced experimental fibrosis. Searching of different databases revealed that transcripts of IGF-I and members of the PDGF/VEGF family are putative miR-29 target sequences. Transfection of Ago-miR-29 did not affect reporter expression of PDGF-B and PDGF-B receptor. However, reporter assays demonstrate functional miR-29 binding to the 3′-UTR of the PDGF-C, IGF-I and VEGF-A mRNA but not to the corresponding mutated sequences taken as negative controls. Furthermore, we were able to show that miR-29 was markedly upregulated after stimulation of HSC with the antifibrogenic mediator HGF. Conclusion: miR-29, that is downregulated during experimental fibrosis, but upregulated by HGF is postulated to be involved in regulation of extracellular matrix synthesis as well as in secretion of the profibrogenic mediators IGF-I, VEGF-A and PDGF-C. Therefore, miR-29, are antifibrogenic miRNAs not only by targeting collagen biosynthesis but also by interfering with the profibrogenic cell communication.