Aline B. Scandurro

Toll‐Like Receptors on Human Mesenchymal Stem Cells Drive Their Migration and Immunomodulating Responses

Adult human bone marrow‐derived mesenchymal stem cells (hMSCs) are under study as therapeutic delivery agents that assist in the repair of damaged tissues. To achieve the desired clinical outcomes for this strategy requires a better understanding of the mechanisms that drive the recruitment, migration, and engraftment of hMSCs to the targeted tissues. It is known that hMSCs are recruited to sites of stress or inflammation to fulfill their repair function. It is recognized that toll‐like receptors (TLRs) mediate stress responses of other bone marrow‐derived cells. This study explored the role of TLRs in mediating stress responses of hMSCs. Accordingly, the presence of TLRs in hMSCs was initially established by reverse transcription‐polymerase chain reaction assays. Flow cytometry and fluorescence immunocytochemical analyses confirmed these findings. The stimulation of hMSCs with TLR agonists led to the activation of downstream signaling pathways, including nuclear factor κB, AKT, and MAPK. Consequently, activation of these pathways triggered the induction and secretion of cytokines, chemokines, and related TLR gene products as established from cDNA array, immunoassay, and cytokine antibody array analyses. Interestingly, the unique patterns of affected genes, cytokines, and chemokines measured identify these receptors as critical players in the clinically established immunomodulation observed for hMSCs. Lastly, hMSC migration was promoted by TLR ligand exposure as demonstrated by transwell migration assays. Conversely, disruption of TLRs by neutralizing TLR antibodies compromised hMSC migration. This study defines a novel TLR‐driven stress and immune modulating response for hMSCs that is critical to consider in the design of stem cell‐based therapies.

The pro-inflammatory peptide LL-37 promotes ovarian tumor progression through recruitment of multipotent mesenchymal stromal cells

Bone marrow-derived mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs) have been shown to engraft into the stroma of several tumor types, where they contribute to tumor progression and metastasis. However, the chemotactic signals mediating MSC migration to tumors remain poorly understood. Previous studies have shown that LL-37 (leucine, leucine-37), the C-terminal peptide of human cationic antimicrobial protein 18, stimulates the migration of various cell types and is overexpressed in ovarian, breast, and lung cancers. Although there is evidence to support a pro-tumorigenic role for LL-37, the function of the peptide in tumors remains unclear. Here, we demonstrate that neutralization of LL-37 in vivo significantly reduces the engraftment of MSCs into ovarian tumor xenografts, resulting in inhibition of tumor growth as well as disruption of the fibrovascular network. Migration and invasion experiments conducted in vitro indicated that the LL-37-mediated migration of MSCs to tumors likely occurs through formyl peptide receptor like-1. To assess the response of MSCs to the LL-37-rich tumor microenvironment, conditioned medium from LL-37-treated MSCs was assessed and found to contain increased levels of several cytokines and pro-angiogenic factors compared with controls, including IL-1 receptor antagonist, IL-6, IL-10, CCL5, VEGF, and matrix metalloproteinase-2. Similarly, Matrigel mixed with LL-37, MSCs, or the combination of the two resulted in a significant number of vascular channels in nude mice. These data indicate that LL-37 facilitates ovarian tumor progression through recruitment of progenitor cell populations to serve as pro-angiogenic factor-expressing tumor stromal cells.

Ovarian cancers overexpress the antimicrobial protein hCAP-18 and its derivative LL-37 increases ovarian cancer cell proliferation and invasion

The role of the pro‐inflammatory peptide, LL‐37, and its pro‐form, human cationic antimicrobial protein 18 (hCAP‐18), in cancer development and progression is poorly understood. In damaged and inflamed tissue, LL‐37 functions as a chemoattractant, mitogen and pro‐angiogenic factor suggesting that the peptide may potentiate tumor progression. The aim of this study was to characterize the distribution of hCAP‐18/LL‐37 in normal and cancerous ovarian tissue and to examine the effects of LL‐37 on ovarian cancer cells. Expression of hCAP‐18/LL‐37 was localized to immune and granulosa cells of normal ovarian tissue. By contrast, ovarian tumors displayed significantly higher levels of hCAP‐18/LL‐37 where expression was observed in tumor and stromal cells. Protein expression was statistically compared to the degree of immune cell infiltration and microvessel density in epithelial‐derived ovarian tumors and a significant correlation was observed for both. It was demonstrated that ovarian tumor tissue lysates and ovarian cancer cell lines express hCAP‐18/LL‐37. Treatment of ovarian cancer cell lines with recombinant LL‐37 stimulated proliferation, chemotaxis, invasion and matrix metalloproteinase expression. These data demonstrate for the first time that hCAP‐18/LL‐37 is significantly overexpressed in ovarian tumors and suggest LL‐37 may contribute to ovarian tumorigenesis through direct stimulation of tumor cells, initiation of angiogenesis and recruitment of immune cells. These data provide further evidence of the existing relationship between pro‐inflammatory molecules and ovarian cancer progression.

Tumors Sound the Alarmin(s)

Recent evidence suggests that inflammatory molecules play critical roles in the development and progression of numerous tumors. However, one specific group of inflammatory molecules whose importance has been established in host immune responses, termed alarmins, has been largely overlooked in cancer biology. The function of several alarmins-including the defensins, LL-37, and HMGB1-in tumor development, progression, or suppression is discussed here. Taken together, these studies indicate that alarmins represent potential new targets for manipulation in a variety of tumors.

NF-κB plays a key role in hypoxia-inducible factor-1–regulated erythropoietin gene expression

OBJECTIVE The aim of this study was to further define the signal transduction pathways leading to hypoxia-inducible factor-1 (HIF-1) erythropoietin (EPO) gene expression. MATERIALS AND METHODS Human hepatocellular carcinoma cells (Hep3B) were exposed to hypoxia (1% oxygen) and examined for mRNA expression, as well as gene transactivation with RT-PCR and luciferase reporter gene assays, respectively. RESULTS Treatment with LY294002 (a selective pharmacological inhibitor of phosphatidylinositol 3-kinase) significantly inhibited EPO protein and mRNA expression in Hep3B cells exposed to hypoxia for 24 hours, while treatment with PD098059 or SB203580 (selective pharmacological inhibitors of the MEK and p38 mitogen-activated protein kinase pathways, respectively) had no significant effects. The activity of AKT, a downstream target of PI3K, was increased by hypoxia and was also inhibited by LY294002. Genetic inhibition of AKT resulted in significant inhibition of NF-kappaB and HIF-1-mediated transactivation, as well as EPO gene expression, in response to hypoxia. Overexpression of constitutively active AKT resulted in increased NF-kappaB and HIF-1 transactivation. The selective inhibitor of NF-kappaB, pyrrolidine dithiocarbamate (PDTC), significantly blocked HIF-1 protein expression. Inhibition of NF-kappaB with a superrepressor dominant negative IkappaBalpha genetic construct also significantly blocked NF-kappaB and HIF-1 transactivation, as well as EPO gene expression. CONCLUSION We propose a key role for NF-kappaB in EPO gene regulation in response to hypoxia.

Leucine Leucine-37 Uses Formyl Peptide Receptor–Like 1 to Activate Signal Transduction Pathways, Stimulate Oncogenic Gene Expression, and Enhance the Invasiveness of Ovarian Cancer Cells

Emerging evidence suggests that the antimicrobial peptide, leucine leucine-37 (LL-37), could play a role in the progression of solid tumors. LL-37 is expressed as the COOH terminus of human cationic antimicrobial protein-18 (hCAP-18) in ovarian, breast, and lung cancers. Previous studies have shown that the addition of LL-37 to various cancer cell lines in vitro stimulates proliferation, migration, and invasion. Similarly, overexpression of hCAP-18/LL-37 in vivo accelerates tumor growth. However, the receptor or receptors through which these processes are mediated have not been thoroughly examined. In the present study, expression of formyl peptide receptor–like 1 (FPRL1) was confirmed on ovarian cancer cells. Proliferation assays indicated that LL-37 does not signal through a G protein–coupled receptor, such as FPRL1, to promote cancer cell growth. By contrast, FPRL1 was required for LL-37–induced invasion through Matrigel. The peptide stimulated mitogen-activated protein kinase and Janus-activated kinase/signal transducers and activators of transcription signaling cascades and led to the significant activation of several transcription factors, through both FPRL1-dependent and FPRL1-independent pathways. Likewise, expression of some LL-37–stimulated genes was attenuated by the inhibition of FPRL1. Increased expression of CXCL10, EGF, and PDGF-BB as well as other soluble factors was confirmed from conditioned medium of LL-37–treated cells. Taken together, these data suggest that LL-37 potentiates a more aggressive behavior from ovarian cancer cells through its interaction with FPRL1. (Mol Cancer Res 2009;7(6):907–15)

Changes in redox affect the activity of erythropoietin RNA binding protein.

We have previously identified a cytosolic protein, erythropoietin RNA binding protein (ERBP), which is up‐regulated in certain tissues in response to hypoxia. To further characterize the interaction of ERBP and erythropoietin (EPO) mRNA, we have examined the role of reduction‐oxidation in the EPO mRNA binding mechanism of ERBP isolated from human hepatoma cells (Hep3B). Reducing agents dithiothreitol (DTT) and 2‐mercaptoethanol (2‐ME) increased ERBP binding activity in a concentration‐dependent manner, whereas the oxidizing agent, diamide, abolished ERBP binding activity. In addition, treatment of Hep3B cell lysates with the irreversible sulfhydryl alkylating agent N‐ethylmaleimide resulted in inhibition of the EPO mRNA‐ERBP complex. Taken together, these findings suggest that sulfhydryl groups may play a role in vivo in the regulation of EPO production through the modulation of ERBP binding activity.

Protein kinase C α protein expression is necessary for sustained erythropoietin production in human hepatocellular carcinoma (Hep3B) cells exposed to hypoxia

Although protein kinase C (PKC) has been implicated as an effector of erythropoietin (EPO) production, its exact role is still uncertain. Hep3B human hepatocellular carcinoma cells were used for this study and were depleted of PKC in three different ways: long-term treatment with phorbol 12-myristate 13-acetate (PMA), selective inhibition with calphostin C, and treatment with PKCalpha antisense oligonucleotides. When EPO-producing Hep3B cells were incubated in 1% O2 (hypoxia) for 24 h, PMA treatment resulted in significant decreases in medium levels of EPO in Hep3B cell cultures at concentrations higher than 10 nM. The specific PKC inhibitor, calphostin C, significantly inhibited medium levels of EPO and EPO mRNA levels in Hep3B cells exposed to 1% O2. Western blot analysis revealed that Hep3B cells express the classical PKCalpha and gamma isoforms, as well as novel PKCepsilon and delta and the atypical zeta isoform. Preincubation with PMA for 6 h specifically down-regulated PKCalpha protein expression. Phosphorothioate modified antisense oligonucleotides specific for PKCalpha also decreased EPO production in Hep3B cells exposed to hypoxia for 20 h when compared to PKCalpha sense treatment. The translocation of PKCalpha from the soluble to particulate fractions was increased in Hep3B cells incubated under hypoxia compared with normoxia (21% O2) controls. These results suggest that the PKCalpha isoform plays an important role in sustaining hypoxia-regulated EPO production.

Redox and Heat Shock Protein Hsp70 Affect the Binding of Erythropoietin RNA Binding Protein to Erythropoietin mRNA

Factors affecting complex formation between the cytosolic protein identified in our laboratory to bind to erythropoietin (EPO) mRNA (ERBP) and this mRNA have been investigated. It was observed that reducing agents, dithiothreitol and 2-mercaptoethanol, enhanced ERBP binding activity in a dose-dependent manner. Oxidizing agents such as diamide abolished complex formation. Treatment with the sulfhydryl alkylating agent N-ethylmaleimide also resulted in inhibition of complex formation, suggesting a possible role of sulfhydryl groups in the interaction between this protein and EPO mRNA.